Atlas of Procedures in Neonatology, 4th Edition

Miscellaneous Sampling

24

Perimortem Sampling

Cynthia J. Tifft

Jayashree Ramasethu

The death of a newborn is devastating for parents and extended families. This is particularly true when the cause of death is a suspected but unconfirmed genetic or metabolic disorder or when the cause of death is unknown. Approximately one third of pediatric admissions have a genetic component to their illness (1), and genetic disease is a major contributing factor in 15% to 25% of all infant deaths (2). Additionally, more than 400 inborn errors of metabolism have been described, and although treatment may be available, many infants die before the diagnosis is made or even considered (3). The majority of inborn errors are autosomal recessive, implying a 25% recurrence risk for future pregnancies. For many of these disorders, arriving at a diagnosis often hinges on timely perinatal and postnatal sampling. Perhaps the greatest service a neonatologist can render to the family of a dying child is appropriate evaluation and timely collection of samples for diagnosis.

During the discussion with the family to withdraw life support or as soon after the infant expires as possible, discuss with the parents the importance of collecting perinatal fluid and tissue samples and performing an autopsy. Postmortem examinations can be crucial in determining cause of death and may have implications for the health of other family members. Such examinations have been shown to alter the recurrence risks for future pregnancies in 22% to 26% of cases (4,5,6 and 7).

If family members are resistant to a complete autopsy, then a limited study with exclusions or permission to obtain tissue samples can also be extremely helpful. Noninvasive techniques such as postmortem magnetic resonance imaging (MRI), especially in conjunction with tissue sampling, may be extremely helpful in arriving at a diagnosis and may be more acceptable to the family (8).

  1. Importance of Perimortem Diagnostic Tests (3,9)
  2. Establish the correct diagnosis.
  3. Give parents an accurate explanation of the cause of death and allay parental fears about what did not cause the child's death.
  4. Permit appropriate testing of asymptomatic parents and siblings, potentially facilitating prenatal diagnosis and preventing mortality and morbidity.
  5. Provide genetic counseling for recurrence risks and discussion of reproductive options and prenatal diagnosis.
  6. Dispel allegations of abuse or neglect.
  7. Indications (10 11,12,13 and 14)
  8. Suspected inborn error of metabolism
  9. Suspected genetic disease or chromosome abnormality
  10. Suspected infectious disease
  11. Unexplained death
  12. Precautions
  13. Informed consent is essential for all procedures.
  14. To prevent bacterial contamination, use sterile procedures even for postmortem sampling.
  15. Perimortem Evaluation
  16. Timely consultation with a clinical and/or biochemical geneticist as soon as a genetic or metabolic disorder is suspected is key in evaluating the neonate and guiding the correct retrieval, collection, and storage of appropriate samples for analysis. When a genetic or biochemical disorder is suspected, the perimortem collection of samples should be considered an emergency procedure (3).
  17. Careful family history including three-generation pedigree. Pay special attention to neonatal deaths or stillbirths, pregnancy losses, and sudden or unexplained infant deaths. Ask about consanguinity, ethnicity, and obtain an obstetric history. Based on the clinical findings, consider work-up for a thrombophilia disorder in the mother (3).
  18. Dysmorphology examination (performed by a clinical geneticist with expertise in dysmorphology if possible)
  19. Document both negative and positive findings.
  20. Pay special attention to:

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  1. Growth parameters (large or small for gestational age)
  2. Distribution, abundance, and texture of the hair
  3. Subtle facial variations including ear configuration and placement and presence of facial or palatal clefts
  4. Eyes for spacing, palpebral fissure length and slant, eyelid malformation, colobomas, microphthalmia, cataracts, or obvious retinal changes
  5. Chest size, sternal or rib malformations, abdominal wall or spinal defects
  6. Digital abnormalities including syndactyly, polydactyly, or positioning defects
  7. Genital or rectal anomalies
  8. Photographs
  9. Obtain photographs of as high a quality as possible.
  10. A fuzzy Polaroid is better than no photo at all.
  11. Obtain both anteroposterior and lateral views.
  12. Obtain close-up photographs of any abnormalities.
  13. Try to obtain photographs without obscuring tubes or tape.
  14. Radiographs
  15. If a skeletal disorder is suspected, radiographs of the infant are of supreme importance and often diagnostic (15).
  16. For small infants, a “babygram” or Faxitron (Faxitron X-ray Corp., Wheeling, IL, USA) study can be used.
  17. Anteroposterior and lateral views should be obtained, as well as hands and feet and any extremity variations.
  18. Magnetic resonance imaging (MR-autopsy) (8)
  19. Advantages
  20. MR is noninvasive and may be acceptable to parents who refuse full autopsy for fear of disfigurement.
  21. High-quality images can be obtained especially of the central nervous system (CNS anomalies are present in 1% of all births) (16), which is often difficult to evaluate by conventional autopsy.
  22. The organs are examined in situ; therefore, complex malformations involving several organs can be evaluated in correct anatomic position. This can be particularly important for CNS and spinal cord anomalies.
  23. MR-autopsy can be performed up to 72 hours after death.
  24. Disadvantages
  25. No tissue samples can be obtained for culture, chromosome, or metabolic analysis.
  26. Complex cardiac malformations and anomalous connections between hollow organs (i.e., tracheo-esophageal fistula or bowel perforations) are difficult to detect by MR-autopsy.
  27. Skeletal malformations may be missed.
  28. Most hospital MR staff are not familiar with examining deceased neonates and will need to be educated about the value of such studies.
  29. Perimortem Sampling (3,10)
  30. Blood samples
  31. Contact the hospital laboratory immediately to save any unused portions of blood specimens sent earlier for other routine tests. Have the serum or plasma separated and frozen and any unused urine or cerebrospinal fluid frozen.
  32. Contact the state laboratory to retrieve any unused portion of blood spots sent for newborn screening. If not previously reported, inquire about the results of the newborn screening.
  33. When death is imminent or the family has elected to withdraw life support measures for an infant, obtain blood samples for further analysis (Table 24.1).
  34. Dried blood spots on filter paper (at least two or three newborn screening cards) stored at room temperature but not in a plastic bag.
  35. Whole blood
  36. 5 mL in lithium heparin tube (separated within 20 minutes of collection and stored at -80°C)
  37. 5 mL in EDTA tube for DNA extraction (can be stored for 48 hours at 4°C prior to extraction)
  38. 5 mL in sodium heparin (can be stored at room temperature or at 4°C overnight for chromosome analysis; do not freeze)
  39. Urine samples (10)
  40. May be used for urine organic acid and amino acid analysis
  41. Collect by catheterization, Crede's method, or bladder tap and freeze immediately. If the sample is contaminated with blood, centrifuge the sample to remove red cells, then freeze. Washing out the bladder with a small amount of sterile saline may yield enough sample for organic acid analysis.
  42. Volume of 5 to 10 mL is ideal, but even the smallest amount of urine available may be useful. As little as 100 µL can be sufficient for organic acid analysis by gas chromatography-mass spectrometry
  43. Hair samples

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  1. Reserve a few strands of hair (plucked from the scalp) for microscopic analysis (17)

TABLE 24.1 Blood Sample Collection

Laboratory Test

Volume (mL)

Tube

Handling

Karyotype

1.5

Sodium heparin

Room temperature or refrigerate, do not freeze

Chromosome microarray analysis temperature

3
3

Sodium heparin
K3 EDTA

Room

Plasma amino acids

1–2

Lithium heparin

On ice; if not processed immediately, separate plasma and freeze

Plasma carnitine

1–3

Lithium heparin

On ice

Very-long-chain fatty acids

3

K3 EDTA

Room temperature

White blood cells for enzymes/DNA

3

K3 EDTA

Room temperature

EDTA, ethylenediaminetetraacetate.

  1. Cerebrospinal fluid
  2. For suspected metabolic disorder, specimen should be obtained prior to death and immediately frozen at -80°C (10,18).
  3. A postmortem sample of cerebrospinal fluid may be obtained by passing a needle through the anterior fontanelle using aseptic technique and may be useful for detecting an infectious disorder (4).
  4. Skin biopsy (19,20)
  5. Skin biopsy specimens may be subjected to microscopic examination or used for culturing skin fibroblasts. Skin fibroblasts can be used for chromosome analysis, for DNA isolation for mutation detection, and for enzyme analysis. Specimens should be obtained as close to the time of death as possible, but skin biopsy up to 2 to 3 days postmortem may still produce a viable culture. In this case, several small biopsies taken from a number of separate sites (skin or fascia) may be more successful.
  6. Clean the inside of the forearm or the anterior thigh with numerous alcohol swabs.
  7. Povidone–iodine should not be used because it can impair cell growth.
  8. Use a 3- or 5-mm punch biopsy or a scalpel to obtain a full thickness of skin. A small piece of sterile fascia (10) or a fine-needle aspiration of tissue (21) may also be used for fibroblast culture.
  9. Place the biopsy tissue in a culture medium such as Hanks' or minimal essential medium (MEM), or a viral transport medium. If these are unavailable, place in sterile saline.
  10. Refrigerate specimen or transport at room temperature. Do not freeze.
  11. Cells can be cultured and then archived in liquid nitrogen for decades and still be successfully recovered for analysis.
  12. Muscle biopsy (10,22,23)
  13. If a neonatal muscular dystrophy or a mitochondrial disorder of energy metabolism is suspected, try to obtain a skeletal muscle biopsy as close to the time of death as possible, but within 2 to 4 hours. A percutaneous or incisional muscle biopsy can be performed at the bedside. A neurologist or surgeon may be more experienced in obtaining an appropriate specimen and/or for providing the muscle clamps necessary to appropriately obtain the specimen.
  14. Incisional biopsy. Make a 2- to 3-cm incision over the quadriceps muscle to expose it. Muscle clamps should be applied prior to removal of the muscle fibers in order to prevent the muscle from contracting. Three muscle fibers should be removed and processed as follows.
  15. Place one on a saline-soaked Telfa pad (Kendall Company, Mansfield, MA, USA) and refrigerate. Do not have the muscle floating in saline. This will be used for light microscopy.
  16. Place a second specimen in 1.5% glutaraldehyde/1% formaldehyde in 0.12 M Sorensen's buffer and place in the refrigerator. If this is not available, place the sample in formalin and leave at room temperature. If neither of these is available, place the specimen on a saline-soaked Telfa pad and refrigerate. This will be used for electron microscopy.
  17. Wrap a third specimen in aluminum foil and snap-freeze in isopentane if available. Alternatively, the specimen can be snap-frozen in liquid

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nitrogen or dry ice and stored at - 80°C. This will be used for enzyme analysis.

  1. Percutaneous biopsy. If an incisional biopsy is not possible, three cores of quadriceps muscle should be obtained percutaneously using a biopsy needle and processed as described above.
  2. Liver biopsy (11,24,25)
  3. If there is hepatic failure or an inborn error of metabolism is suspected, try to obtain a biopsy of the liver as close to the time of death as possible. If the autopsy will not be performed for several hours and the hospital pathologist concurs, or if the family has declined autopsy but permitted tissues to be obtained, an open wedge biopsy or percutaneous biopsy can be performed at the bedside. If the biopsy has to be delayed for any reason, specimens obtained for biochemical analysis even up to 72 hours after the time of death may provide significant results (11).
  4. Wedge biopsy. Make a 2-cm skin incision just below the right costal margin. With forceps, lift up the right lobe of the liver, and with a clean scalpel, remove a wedge. Cut the wedge into several 5-mm cubes and place in a plastic cryotube. Snap-freeze the tube in liquid nitrogen, or if this is not available, bury it in dry ice. Store at 280°C
  5. Percutaneous biopsy. A 16- or 18-gauge Menghini needle (Allegiance Healthcare Corp., McGraw Park, IL, USA), a Jamshidi needle (Pharmaseal; Baxter Healthcare Corp., Valencia, CA, USA), a 16-gauge Klatskin needle (Becton Dickinson, Franklin Lakes, NJ, USA), a Tru-Cut needle (Travenol Laboratories, Deerfield, IL, USA), or a spring-loaded disposable liver biopsy needle (Monopty; Bard Peripheral Technologies, Covington, GA, USA) may be used. With the infant positioned supine, mark a site slightly below the right midaxillary line, at the ninth or tenth rib. Make an incision in the skin 1/8 in long with a no. 11 scalpel blade above the rib. Attach a locking 10-mL syringe filled with 5 mL of normal saline to the needle. Flush to remove the air and direct the liver biopsy needle through the skin incision, parallel to the surface the infant is on, directed toward the xiphoid, nipple, or opposite shoulder. Flush with 1 mL of saline to clear the cannula. Advance 2 to 3 cm while creating negative pressure in the syringe by pulling back the plunger 1 to 2 mL and turning it into the locking position. Withdraw the needle and syringe assembly. If the automatic spring-loaded system is used, negative pressure is not required. Several cores of liver should be obtained using a biopsy needle and processed as described above.
  6. Placenta and umbilical cord
  7. Ensure that the placenta is sent to the pathologist for evaluation for every sick infant (26).
  8. Examination of the placenta may provide clues to maternal or fetal vascular problems (27), in utero infectious or abnormal inflammatory responses (28), or lysosomal storage disorders (29).
  9. A sample of the umbilical stump in the newborn may identify invasive inflammation in cases of sepsis.
  10. The Postmortem Professional and Family Conference (8,30)
  11. It is important to keep in contact with the family. Apprise them of what studies are to be performed on their deceased infant and how long they will take, and what samples remained stored if further diagnostic testing becomes available.
  12. If not done previously, obtain a complete three-generation family history as described in D.2. Obtain the services of a geneticist or genetic counselor in reviewing the family history and assigning recurrence risks.
  13. Review the prenatal history for any exposures to infection or potential teratogens including environmental toxins. Remember, the parents are looking for answers and will usually be eager to share any bit of information they think might be helpful in arriving at a cause of death.
  14. If possible, schedule a multidisciplinary conference involving the neonatologists as well as the clinical or biochemical geneticist, pathologist, radiologist, or other subspecialists who cared for the child or who have taken part in the postmortem evaluation. Carefully review the findings and arrive at a diagnosis or possible diagnoses, including recurrence risks and reproductive options available to the couple. Identify any family members who may be asymptomatic carriers or at risk for the disorder so that additional testing can be offered.
  15. When the autopsy and any special studies are complete, schedule a conference with the family. Review the findings. It is often important to explain what did not lead to the infant's death as well as the suspected or confirmed final diagnosis. With the help of a geneticist or genetic counselor, discuss the recurrence risks for future pregnancies and what types of studies (e.g., chromosome or biochemical analysis, ultrasound, MRI, or

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fetal echocardiography) could be used to monitor subsequent pregnancies. If available, offer testing to family members who are suspected carriers or who may be asymptomatic but at risk for the disorder (e.g., the deceased child's siblings).

  1. Provide the family and the referring physician with a copy of the autopsy report and any diagnostic testing results for their records. Provide a letter summarizing the postmortem conference. These may become very important to them in the future.
  2. Finally, remain available to the family to answer any additional questions or concerns that may arise over the next several weeks to months as they grieve and come to accept of the loss of their child.

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