MISCELLANEOUS SURGICAL PATHOLOGY

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CHAPTER 19 MISCELLANEOUS SURGICAL PATHOLOGY

Pathology of pediatric hair samples
Blood film examination for vacuolated lymphocytes

PATHOLOGY OF PEDIATRIC HAIR SAMPLES

Introduction (Smith et al 2005)

• Hair shafts may be examined using routine light microscopy
• Wide range of morphological abnormalities (Figs 19.1, 19.2)

image many associated with specific underlying disorders
• Several important clinical groups of pediatric patients where light microscopic examination of hair may provide valuable diagnostic information

image erythrodermic infant with immunodeficiency

– trichorrhexis invaginata with Netherton’s syndrome

image neurologically impaired child

– pili torti with Menkes disease
– tiger tail anomaly with trichothiodystrophy

image immunological/hematological disorders and ectodermal dysplasias may also exhibit abnormal hair appearances

• Around half of samples for these indications will show some abnormality
image

Fig. 19.1

image

Figs 19.1–19.2 Photomicrographs of hair shafts showing patterns of normal variation.

Types of sample and handling

(Garn 1951, Leblond 1951, Eilers 1982, Shelley 1983, Caserio 1987, Crounse 1987, De Berker 1997, Smith et al 2005)

• Shaft abnormalities are best seen in cut samples

image plucking may cause unwanted breakages, especially with trichorrhexis, making the sample non-diagnostic

• Follicle disorders (such as loose anagen syndrome) can only be diagnosed on a sample of plucked hair containing the root

image misshapen anagen hairs

– lack external root sheaths
– bulbs distorted
– cuticle just above the root has a ‘wrinkled stocking’ appearance

· above this zone the rest of the hair shaft appears normal

• Plucking carried out either by:

image gripping less than 10 hairs between finger and thumb, or
image gripping a couple of rows of hairs with a needle holder at the base

– and pulling sharply

• Cut hairs should be cut close to the scalp to allow examination of the shaft
• Hairs are usually dry mounted without medium

image placed into a rectangular frame with double sided sticky tape edges on a microscope slide
image hairs lined up in parallel

– securing ends to the frame

image coverglass placed over the frame

• In cases examined for pigment abnormalities (Chediak–Higashi or Griscelli)

image use a mountant (DPX)

• Examine as many strands as possible

image not every hair may demonstrate morphological abnormalities in the lengths examined

Histopathological features

Normal hair

• Normal hair shafts are cylindrical

image smooth surface
image varying diameters
image ovoid or round profiles in cross section

• Normal hair pigment within the shaft is uniformly distributed

Types of morphological abnormality

• Wide range of morphological abnormalities may be seen including:

image non-specific changes:

– weathering

· lifting and damage of cuticular cells

– splits

· especially distal shaft

– twisting (but not classical pili torti)
– mild flattening or grooving

image pili torti (Fig 19.3)

– twists completely rotated through 180° around their long axis at irregular intervals
image trichorrhexis nodosa (Figs 19.4, 19.5)

– swellings along the shafts associated with areas of breakage
– indicates that the hair is easily breakable as a result of injury
image trichorrhexis invaginata (Fig 19.6) (Ito et al 1984)

– nodular swellings with one part apparently indenting the ‘cupped’ other portion

· resembles a bamboo shoot

image pili canaliculati et trianguli

– longitudinal canalicular depressions with kidney shaped or triangular cross sections

image pigment clumping or hypopigmentation
image ‘tiger tail’ appearance under polarized light (Fig 19.7)
image root abnormalities

image

Fig 19.3 Photomicrograph of hair shaft showing pili torti with flattening and twists completely rotated through 180° around the long axis at irregular intervals.

image

Fig. 19.4

image

Figs 19.4–19.5 Photomicrographs of hair shafts showing trichorrhexis nodosa, with areas of shaft breakage.

image

Fig 19.6 Photomicrograph of hair shaft showing trichorrhexis invaginata, with nodular swellings and ‘cup’ appearance, in Netherton syndrome.

image

Fig 19.7 Photomicrograph of hair shaft showing banding under polarized light; tiger-tail anomaly.

Associations with specific diagnoses

• Around 25% of cases submitted have abnormalities compatible with specific diagnoses
• Menkes disease (Williams et al 1978, Martin et al 1978)

image pili torti (Fig 19.8)

– Xq12-q13 ATP7

· encodes ATPase Cu2+ transporting molecule

• Netherton’s syndrome

image trichorrhexis invaginata

– autosomal recessive
– mutations in SPINK5 gene (Descargues et al 2005)

· encodes serine protease inhibitor
· wide range of mutations

• Trichothiodystrophy (Hansen et al 1993, Liang et al 2005)

image ’tiger tail’ anomaly

– 19q13.2-q13.3 ERCC2/XPD

· encode helicase subunits of transcription/repair vector TFIIH

• Griscelli syndrome

image 15q21 MYO5A/RAB27A (Pastural et al 2000, Menasche et al 2003, Celik et al 2007)
image pigment clumping

• Chediak–Higashi syndrome (Figs 19.9, 19.10)

image 1q42.2 Lysosomal trafficking regulator gene (LYST/CHS)
image pigment clumping

– Chediak–Higashi and Griscelli syndromes show similar features

· also examine a blood film: abnormal neutrophil granulation in Chediak–Higashi

• Monilethrix (Fig 19.11)

image 12q13 Keratin genes
image beading as a result of periodic narrowing of the shaft
• Uncombable hair syndrome (Hicks et al 2001)

image pili canaliculati et trianguli
• Loose anagen syndrome (Hamm et al 1989)

image absent inner or outer root sheath and a wrinkled stocking effect
image

Fig 19.8 Photomicrograph of hair shafts showing pili abnormal twisted hair.

image

Fig 19.9 Photomicrograph of hair shafts showing abnormal pigment clumping in Chediak–Higashi syndrome.

image

Fig 19.10 Photomicrograph of a neutrophil in a blood film showing abnormal granulation in Chediak–Higashi syndrome.

image

Fig 19.11 Photomicrograph of a hair shaft under polarized light showing monilethrix, with beading and narrowing.

BLOOD FILM EXAMINATION FOR VACUOLATED LYMPHOCYTES

Introduction (Anderson et al 2005)

• Wide range of metabolic diseases may result in abnormal accumulation of metabolic byproducts and abnormal cytoplasmic vacuolation of lymphocytes

image identifiable on routine blood film examination

– rapid and cheap screening test

• These diseases include:

image Batten’s disease (neuronal ceroid lipofuscinosis)
image Salla disease
image I cell disease
image B-galactosidase deficiency (GM1 gangliosidosis)
image Mucopolysaccharidoses
image Niemann–Pick disease
image Fucosidosis
image Mannosidosis
image Wolman’s disease
image Glycogenoses

• In cases in which vacuolated lymphocytes are detected

image further investigation directed by specific clinical features

– confirmation of the diagnosis usually requires enzyme analysis or other specific tests

image most common indications are:

– developmental delay/regression
– ataxia
– seizures
– cardiomyopathy

• In cases with these indications, when used as a screening test around 5% of cases show vacuolated lymphocytes

Sample preparation and examination

• Routine blood film prepared (EDTA sample)

image if EM also needed, a buffy coat sample is also required
image routine May-Grunwald–Giemsa staining

• Examine ‘thin’ end of film near the tail, for lymphocyte cytoplasmic vacuolation (Figs 19.12–19.15)

image at least 100 lymphocytes should be examined

– vacuolation may vary in extent with age and disease

• Caution not to erroneously interpret basophil vacuolation as abnormal lymphocyte vacuolation
image

Fig 19.12 Photograph of a blood film showing the correct area near the ‘tail’ of the film, for examination for vacuolated lymphocytes.

image

Fig 19.13 Photomicrograph of a buffy coat demonstrating the layers of cellular components including platelets, white cells and red cells.

image

Fig 19.14 Photomicrograph of a buffy coat demonstrating vacuolated lymphocytes.

image

Fig 19.15 Photomicrograph of a blood film showing a range of lymphocyte vacuolation. (A) Routine May-Grunwald–Giemsa stained blood film showing two lymphocytes with many large bold vacuoles, such as are seen in GM1 gangliosidosis, and juvenile Batten’s disease. (B) Routine May-Grunwald–Giemsa stained blood film showing a lymphocyte with discrete small vacuoles seen in Pompe’s disease and adult acid maltase deficiency. (C) Periodic acid–Schiff (PAS) stained blood film showing a lymphocyte with PAS positive inclusions in Pompe’s disease and adult acid maltase deficiency. (D) Toluidine blue stained blood film showing metachromatic cytoplasmic inclusions in a lymphocyte. (E) Enzyme histochemical demonstration of β-galactosidase in a blood film showing normal enzyme activity in a lymphocyte and eosinophil but negative activity in a neutrophil. (F) Enzyme histochemical demonstration of β-galactosidase in a blood film showing absent enzyme activity in a lymphocyte and a granulocyte seen in GM1 gangliosidosis and galactosialidosis.

Histopathological features

• Examine film for lymphocyte vacuolation

image presence or absence of vacuolated lymphocytes
image characteristics of vacuolation

• Acid maltase deficiency (infantile Pompe’s disease or adult acid maltase deficiency)

image 17q25.2-q25.3

– confirmation by enzyme assay on cultured fibroblasts
– 1–6 small distinct cytoplasmic vacuoles

image stain strongly with periodic acid–Schiff after celloidin protection of the highly soluble form of the stored glycogen

• Salla disease (sialic acid storage disease) (Aula et al 1979)

image sialin (sialic acid transporter protein) deficiency

– 6q14-q15; mutations in SLC17A5

image numerous small vacuoles
image confirmation by urine analysis for sialic acid
• Sialidosis type 2

image neuraminidase deficiency

– 6p21.3

image numerous large bold vacuoles
image confirmation by enzyme assay of cultured fibroblasts

• Galactosialidosis (neuraminidase deficiency with a galactosidase deficiency)

image neuraminidase and galactosidase deficiency secondary to cathepsin A/ lysosomal protective protein deficiency

– 20q13.1

image numerous large bold vacuoles
image confirmation by enzyme assay of cultured fibroblasts

• I cell disease (mucolipidosis II) (Terashima et al 1975)

image N-Acetyl glucosaminyl-phosphotransferase deficiency

– 4q21-23

image numerous large bold vacuoles
image confirmation by isoscreen of plasma for aryl sulfatase A and fibroblast culture
• GM1 gangliosidosis (Giugliani et al 1985)

image β-galactosidase deficiency

– 3p21.33

image numerous large bold vacuoles
image confirmation by enzyme assay of white blood cells and fibroblasts and histochemical β-galactosidase detection on blood films

– absent in lymphocytes and neutrophils

• Mucopolysaccharidosis 1H (Hurler)

image A-L-Idurinidase deficiency

– 4p16.3

image occasional vacuoles in some lymphocytes

– some with basophilic inclusions (Gasser cells)

image confirmation by enzyme assay on white blood cells and urinary glycosaminoglycans

• Mucopolysaccharidosis 1S (Scheie), MPS 1H/S (Hurler–Scheie), MPS 2 (Hunter)

image iduronate sulfatase deficiency

– Xq28

image occasional vacuoles in some lymphocytes

– metachromatic inclusions

image confirmation by enzyme assay on white blood cells and urinary glycosaminoglycans

• Mucopolysaccharidosis 3 (San filippo)

image heparan sulfate sulfatase

– 17q25.3

image occasional vacuoles in some lymphocytes

– metachromatic inclusions

image confirmation by enzyme assay on white blood cells and urinary glycosaminoglycans

• Mucopolysaccharidoses (other)

image range from no vacuoles through to small vacuoles in many lymphocytes
image confirmation by enzyme assay on white blood cells and urinary glycosaminoglycans

• Niemann–Pick A

image sphingomyelinase deficiency

– 11p15.4-p15.1

image 1–6 small vacuoles in most lymphocytes
image confirmation by enzyme assay of white blood cells

• Fucosidosis

image A-L-Fucosidase deficiency

– 1p34

image small discrete vacuoles in lymphocytes
image confirmation by enzyme assay of white blood cells

• Juvenile subtype of Batten’s disease (neuronal ceroid lipofuscinosis type 3; NCL3) (Kimura and Goebel 1988, Lake and Cavanagh 1978)

image enzyme defect not known

– 16p12.1

image numerous large distinct cytoplasmic vacuoles

– no specific staining features

image confirmation by EM of blood or skin showing fingerprint inclusions

– ultrastructural examination of buffy coat may allow further specific diagnosis of Battens disease types:

· classic infantile type: granular osmophilic deposits (GRODs)
· late infantile type: curvilinear bodies
· juvenile type: fingerprint inclusions

• Mannosidosis

image α-Mannosidase deficiency

– 19cen-q12

image variable

– numerous small discrete to several large bold vacuoles

image confirmation by enzyme assay of white blood cells

• Wolman’s disease

image acid esterase deficiency (acid lipase, acid cholesterol ester hydrolase)

– 10q24-q25

image 1–6 small discrete vacuoles in most lymphocytes

– stain positive with oil red O or Sudan black

image Confirmation by enzyme assay of white blood cells

All figures in this chapter are from Anderson et al 2005 and Smith et al 2005.

REFERENCES

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Caserio RJ. Diagnostic techniques for hair disorders. Part I: Microscopic examination of the hair shaft. Cutis. 1987;40:265-270.

Caserio RJ. Diagnostic techniques for hair disorders. Part II: Microscopic examination of hair bulbs, tips, and casts. Cutis. 1987;40:321-325.

Celik HH, Tore H, Tunali S, Tatar I, Aldur MM. Light and scanning electron microscopic examination of hair in Griscelli syndrome. Saudi Med J. 2007;28:1275-1277.

Crounse RG. The diagnostic value of microscopic examination of human hair. Arch Pathol Lab Med. 1987;111:700-702.

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Descargues P, Deraison C, Bonnart C, et al. Spink5-deficient mice mimic Netherton syndrome through degradation of desmoglein 1 by epidermal protease hyperactivity. Nat Genet. 2005;37:56-65.

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Garn SM. The examination of hair under the polarizing microscope. Ann. NY Acad Sci. 1951;53:649-652.

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Pastural E, Ersoy F, Yalman N, et al. Two genes are responsible for Griscelli syndrome at the same 15q21 locus. Genomics. 2000;63:299-306.

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