1. TINJAUAN PUSTAKA IMUNOLOGI
ASPEK KLINIS DAN LABORATORIS
(SEROLOGIS) PADA INFEKSI
VIRUS HERPES SIMPLEK
Oleh:
Binawati / Endang Retnowati
1
2. PENDAHULUAN (1)
• Infeksi Herpes simplex disebabkan oleh
herpes simplex virus (HSV)
• HSV ada dua tipe :
1. HSV-1 herpes labialis
2. HSV-2 herpes genitalis
2
3. PENDAHULUAN (2)
• Infeksi HSV menjadi masalah karena:
1. transmisi virus dapat terjadi dari
penderita yang asimtomatik,
2. pengaruhnya terhadap kehamilan dan
bayi / janin dalam kandungan,
3. pengaruh pada penderita
imunokompromais,
4. dampak kejiwaan,
5. serta kemungkinan timbulnya resistensi
virus
3
4. HSV (1)
• HSV merupakan virus DNA anggota dari
famili Herpesviridae
• HSV ds (double stranded) DNA
enveloped viruses
• Virion terdiri dari empat struktur yaitu
envelope, tegumen, nucleocapsid dan DNA
yang berisi inti (core)
• Genom virus terbentuk dari protein
ikosahedral
4
6. HSV (3)
• Struktur genom dari kedua subtipe sama
dan derajad homologinya 50-70%
• HSV-1 dan HSV-2 masing-masing memiliki
region spesifik yang digunakan untuk
membedakan kedua tipe HSV
• Perbedaan kedua tipe terletak pada
komposisi molekuler genomnya dan
terefleksi pada struktur glikoprotein dari
peptidanya.
6
7. PATOGENESIS (1)
• Permukaan mukosa atau bagian kulit yang
mengalami abrasi :
- tempat masuknya virus HSV
- tempat multiplikasi virus
• infeksi primer : infeksi pada seseorang
yang sebelumnya belum pernah terinfeksi
dengan HSV-1 atau HSV-2 (seronegatif)
• Respon humoral pada tahap awal meliputi
Ig M yang bersifat sementara dan Ig G
yang bersifat menetap
7
8. PATOGENESIS (2)
• Setelah virus masuk replikasi (sel
epidermis dan dermis) fusi, nucleocapsid
masuk virus dilepaskan dari virion
DNA virus yang tereplikasi tersebut,
selanjutnya dipaket dalam capsid yang
diberi envelope pada membran dalam dari
inti sel inang ditransportasi melalui
aparatus golgi ke bagian ekstraselular
• Dari sel epitel HSV dapat menginfeksi
syaraf sensoris atau otonomik regional,
dan menyebar melalui axon syaraf menuju
ke neuron
8
9. PATOGENESIS (3)
• Keadaan laten : genom virus terpelihara
dalam keadaan represi oleh sel normal
dan tidak menimbulkan efek pada neuron
inang
• Reaktivasi : virus yang keluar dari neuron
ke sel epitel menyebabkan replikasi virus
dan penampakan kembali virus pada
permukaan mukosa
• Kedua mekanisme infeksi laten dan
reaktivasi HSV belum diketahui secara
jelas
9
10. PATOGENESIS (4)
Gambar 1 : Establishment dan reaktivasi infeksi laten virus herpes
(diambil dari animal viruses) 10
11. PATOGENESIS (5)
• Beberapa pencetus yang memicu
terjadinya reaktivasi :
1. stress fisik atau stress psikis,
2. infeksi pneumokokus,
3. infeksi meningokokus,
4. panas,
5. irradiasi, termasuk sinar matahari,
6. menstruasi.
• Infeksi HSV pada otak terutama melalui
transmisi neuronal yang berasal dari
syaraf trigeminal atau olfaktorius
11
12. PATOGENESIS (6)
• Daerah otak yang paling sering terinfeksi
HSV adalah lobus temporalis media dan
lobus frontalis inferior
• Kerusakan jaringan syaraf otak
disebabkan destruksi langsung oleh virus
atau tidak langsung melalui mekanisme
imunologis
12
13. MANIFESTASI KLINIS (1)
• Ada tiga episode herpes :
1. episode primer / infeksi primer
2. infeksi reaktivasi
3. episode pertama infeksi non primer
• Infeksi pada satu daerah tidak dapat
mencegah daerah lain terhadap infeksi
berikutnya
• Infeksi berulang biasanya berlokasi pada
atau dekat infeksi primer
13
20. PEMERIKSAAN SEROLOGI HSV (2)
• Gold standard deteksi antibodi terhadap HSV
western blot (WB)
• Indikasi pemeriksaan imuoasai HSV :
1. konfirmasi adanya infeksi primer
2. kasus dicurigai ensefalitis karena HSV
3. penderita immunosuppressed dan unknown
febris lama dengan penyebab belum diketahui
4. bayi dengan kelainan kongenital yang tidak
diketahui penyebabnya
5. skrining kesehatan (penderita routine sexual)
6. ibu hamil atau suaminya dicurigai menderita
HSV genital
20
21. PEMERIKSAAN SEROLOGI HSV (3)
Gambar 6 : Alur pemeriksaan HSV-1 dan HSV-2 pada wanita
hamil dan pasangannya 21
23. PEMERIKSAAN SEROLOGI HSV (5)
• IgM spesifik HSV tidak membantu
diagnosis infeksi primer karena IgM HSV
dapat ditemukan pada reaktivasi
• Pada infeksi virus, pemeriksaan IgG
avidity spesifik untuk mengetahui
infeksi primer atau infeksi lampau
• Hasil IgG avidity spesifik :
- rendah infeksi primer
- tinggi infeksi lampau atau rekuren
23
24. PEMERIKSAAN SEROLOGI HSV (6)
IMUNOASAI ENZIM (EIA)
• Prinsip dasar dari EIA : ELISA tidak
langsung (indirect)
• Keuntungan : sensitif, praktis dan cepat
• Kerugian : dibutuhkan pengalamanan
yang cukup untuk mengkonstruksi ELISA
• Uji ELISA tidak spesifik kecuali dipakai
glikoprotein G1 (gG1) dan gG2 sebagai
antigen
24
25. PEMERIKSAAN SEROLOGI HSV (7)
UJI HEMAGLUTINASI TAK LANGSUNG
(IHA)
• Prinsip dasar : SDM domba yang
disensitisasi antigen HSV bila direaksikan
dengan serum penderita (mengandung
antibodi terhadap HSV) aglutinasi
• Keunggulan IHA :
- hasil diperoleh dalam satu hari
- dapat melacak antibodi yang baru
diproduksi pada infeksi primer maupun
antibodi stabil pada infeksi laten, dan
menahun
25
26. PEMERIKSAAN SEROLOGI HSV (8)
UJI HAMBATAN HEMAGGLUTINASI (IHA
INHIBITION)
• Prinsip dasar:didasarkan kemampuan
antigen homolog menghambat antibodi
secara lengkap dan antigen yang
heterolog hanya memberikan hambatan
parsial
• Kekemahan : baik antigen IHA maupun
SDM domba yang disensitisasi harus
diproduksi secara lokal
26
27. PEMERIKSAAN SEROLOGI HSV (9)
Penentuan tipe HSV antisera dengan uji hambatan IHA :
IHA dengan sel yang IHA dengan sel yang
Tipe Ab HSV tersensitisasi HSV-1 tersensitisasi HSV-2
setelah absorbsi serum setelah absorbsi serum
dengan dengan
HSV-1 HSV-2 Kontrol HSV-1 HSV-2 Kontrol
1 O + + O O +
2 O O + + O +
1 dan 2 O + + + O +
Tipe tidak tentu O O + O O +
+ = aglutinasi (penurunan titer kurang dari 4 kali dibandingkan kontrol)
O = hambatan (penurunan titer lebih dari 4 kali dibandingkan kontrol)
27
28. INTERPRETASI HASIL (1)
• Kasus dicurigai infeksi primer
konfirmasinya diperiksa interval 10 hari-3
minggu
• Imunoasai antibodi HSV tidak banyak
berguna pada infeksi berulang
• Kasus dicurigai ensefalitis HSV antibodi
dalam CSF 6% diatas kadarnya dalam
darah, berarti amat besar kemungkinan
adanya produksi lokal antibodi dan infeksi
SSP yang baru terjadi
28
29. INTERPRETASI HASIL (2)
• Bayi dengan kelainan kongenital belum
jelas penyebabnya penentuan IgM anti-
HSV, mengkonfirmasi atau menyingkirkan
HSV sebagai penyebabnya
• Risiko penularan pada bayi amat besar
dari ibu hamil (sero-HSV yang negatif)
kenaikan titer IgG 4 kali dengan interval
10-21 hari, perlu tindakan
29
30. INTERPRETASI HASIL (3)
• Hasil IgG dan IgM positif dengan aviditas
IgG yang rendah, menunjukkan bahwa
adanya infeksi terjadi kurang dari empat
bulan
• Hasil IgG dan IgM positif dengan aviditas
IgG yang tinggi menunjukkan infeksi
terjadi lebih dari empat bulan
30
31. INTERPRETASI HASIL (4)
Tabel 5 : Klasifikasi infeksi HSV genital berdasar klinis, virologi dan serologi
Detection of HSV antibodies
Clinical Type of virus Acute phase serum Convalescent phase Classification of
designa isolation serum infection
tion
First HSV-2 None HSV-2 Primary HSV-2
episode HSV-1 None HSV-1 Primary HSV-1
HSV-2 HSV-1 HSV-1 and HSV-2 Nonprimary HSV-2
HSV-1 HSV-2 HSV-1 and HSV-2 Nonprimary HSV-1
HSV-2 HSV-2 with or HSV-2 with or First symptoms of
without HSV-1 without HSV-1 prior HSV-2
infection ;
recurrent HSV-
2
HSV-2 HSV-2 with or HSV-2 with or recurrent HSV-2
without HSV-1 without HSV-1
HSV-1 HSV-1 with or HSV-1 with or recurrent HSV-1
without HSV-2 without HSV-2
31
33. MANIFESTASI KLINIS
INFEKSI ORAL-FASIAL
• Tanda dan gejala klinis hari ke 3-14,
meliputi panas, malaise, mialgia, malas
makan, irritabilitas, dan adenopati servikal
• Sebelum terjadi lesi peningkatan
sensitivitas, kesemutan dan rasa terbakar
ringan
• Reaktivasi HSV ganglia trigeminal
• 50-70% penderita seropositif mengalami
dekompresi trigeminal nerve root dan 10-
15% penderita terinfeksi setelah kira-kira
3 hari post ekstraksi gigi 33
34. MANIFESTASI KLINIS
INFEKSI GENITAL
• Herpes genital primer episode pertama
ditandai dengan panas, pusing, dan
mialgia
• gejala lokal yang menonjol : nyeri,
itching, disuria, urethral dan vaginal
discharge serta limfadenopati inguinal
• Tingkatan lesi dapat bervariasi, meliputi
vesikel, pustula, atau ulkus eritema yang
sangat nyeri
34
35. MANIFESTASI KLINIS
INFEKSI SSP DAN PERIFER
• HSV ensefalitis yang khas penyakit akut
dengan gejala serebral umum dan fokal
• Gejala prodromal : malaise, demam dan
mual
• Gejala ensefalopati : letargi, kebingungan
dan delirium
• Kejang kejang umum atau kejang fokal
• HSV ensefalitis harus dibedakan dengan
meningitis aseptik herpes simplex, yang
terjadi bersamaan dengan infeksi genital
HSV-2
35
36. MANIFESTASI KLINIS
• Metode paling sensitif dan non invasif
untuk diagnosis dini HSV ensefalitis
pemeriksaan HSV DNA pada cairan
serebrospinal dengan PCR
• Adanya HSV antibodi dalam CSF dan HSV
DNA yang persisten dalam CSF dapat
membantu menegakkan diagnosis
36
37. MANIFESTASI KLINIS
INFEKSI HSV NEONATAL
• Populasi yang terinfeksi HSV neonatus
(bayi <6 bulan) mempunyai frekuensi
kejadian infeksi pada viseral dan / atau
CNS paling tinggi
• Angka kematian herpes neonatal 65%,
<10% neonatus dengan infeksi CNS
berkembang normal
• Manifestasi dibagi :
1. Infeksi lokal pada kulit, mata dan mulut
2. Infeksi lokal SSP
3. Infeksi diseminata 37
38. MANIFESTASI KLINIS
Tabel 3 : Faktor risiko kejadian morbiditas dan mortalitas
pada infeksi HSV neonatus
38
40. MANIFESTASI KLINIS
• Risiko terkena herpes neonatal pada :
- wanita HSV-1 seropositif (awal hamil)
1/3800
- wanita HSV-2 seropositif 1/4600
karena ibu HSV-2 seropositif mengimunisasi
janinnya secara transplasenta dengan IgG anti
HSV-2 dan bayi dilahirkan cara operasi caesar
diperlukan pendekatan preventif dengan
pemeriksaan serologi selama kehamilan
40
43. Testing with symptoms: viral culture
If symptoms of herpes appear, they can vary widely from
person to person. If a person does experience signs of
infection, we recommend obtaining a culture test (a swab
from the symptom) within the first 48 hours after a lesion
appears. Results are usually available in about a week's
time.
The major advantage of the culture is its accuracy in giving
a positive result. A culture can also be “typed” to determine
whether the infection is caused by HSV-1 or HSV-2. If you
test positive by viral culture, you can be sure you have the
virus.
The major disadvantage of the culture is its high rate of
false negatives. Because a culture works by requiring virus
that is active, if a lesion is very small, or is already
beginning to heal, there may not be enough virus present
for an accurate culture. Beyond 48 hours of the symptoms
appearing, there is a risk of receiving a false negative test
result. Viral culture is even less accurate during recurrences
(positive in only about 30% of recurrent outbreaks).
43
44. • When an individual contracts herpes, the
immune system responds by developing
antibodies to fight the virus: IgG and IgM.
Blood tests can look for and detect these
antibodies, as the virus itself is not in
blood. IgG appears soon after infection
and stays in the blood for life. IgM is
actually the first antibody that appears
after infection, but it may disappear
thereafter.
44
45. IgM tests are not recommended because of three
serious problems:
1. Many assume that if a test discovers IgM, they have
recently acquired herpes. However, research shows that
IgM can reappear in blood tests in up to a third of people
during recurrences, while it will be negative in up to half of
persons who recently acquired herpes but have culture-
document first episodes. Therefore, IgM tests can lead to
deceptive test results, as well as false assumptions about
how and when a person actually acquired HSV.
2. 2. In addition, IgM tests cannot accurately distinguish
between HSV-1 and HSV-2 antibodies, and thus very easily
provide a false positive result for HSV-2. This is important
in that most of the adult population in the U.S. already has
antibodies to HSV-1, the primary cause of oral herpes. A
person who only has HSV-1 may receive a false positive for
HSV-2.
3. 3. IgM tests sometimes cross-react with other viruses in
the same family, such as varicella zoster virus (VZV) which
causes chickenpox or cytomegalovirus (CMV) which causes
mono, meaning that positive results may be misleading.
45
47. DIA-
DIA- HSV 1/2-IgG and DIA- HSV 2-IgG
1/2- DIA- 2-
• Principle of the method: two stage ELISA based on «IgM-
cupture» principle
• Clinical materials: human serum or plasma
• DIA-HSV ½-IgM: Detection of specific IgM antibodies to
herpes simplex virus 1 and 2 types
DIA-HSV 2-IgM: Detection of specific IgM antibodies to
herpes simplex virus 2 type
• Immunosorbent – monoclonal antibodies to human IgM
• Conjugate:
DIA-HSV ½-IgM: specific purified recombinant antigens
gG1 HSV1 and gG2 HSV2 conjugated with horseradish
peroxidase;
DIA-HSV 2-IgM: specific purified recombinant antigen of
herpes simplex virus 2 (gG2) conjugated with horseradish
peroxidase
• Packing configuration: strip (lockwell) microplate
TMB chromogen
96 tests
• Incubation time: 2 hours
• Shelf life: 12 months
47
48. DIA-
DIA- HSV 1/2-IgG and DIA- HSV 2-IgG
1/2- DIA- 2-
Assay principle
• wells coated with antigens of herpes
simplex virus 1 and 2
• adding of sera and controls
• incubation for 60 minutes, 37°С
• adding of conjugate
• incubation for 30 minutes, 37°С
• adding of chromogen
• incubation for 30 minutes, 18-25°С
• reaction termination
• result reading
48
50. DIA-
DIA-HSV 1/2-IgМ and DIA-HSV 2-IgM
1/2- DIA- 2-
• Principle of the method: two stage ELISA
procedure based on «IgM-capture» principle
• Clinical materials: human serum or plasma
• Detection of specific to Rubella virus IgM
antibodies
• Immunosorbent – monoclonal antibodies to
human IgM
• Conjugate – purified antigen of Rubella virus
conjugated with horseradish peroxidase
• Packing configuration: strip (lockwell) microplate
TMB chromogen
96 tests
• Incubation time: 1,5 hours
• Shelf life: 12 months 50
51. DIA-
DIA-HSV 1/2-IgМ and DIA-HSV 2-IgM
1/2- DIA- 2-
• Assay principle
• wells coated with monoclonal antibodies to
human IgM
• adding of sera incubation for 60 minutes,
37°С
• adding of conjugate
• incubation for 30 minutes, 37°С
• adding of chromogen
• incubation for 30 minutes, 18-25 °С
• reaction termination
• result reading
51
52. CYTOLOGY TESTS
In the past, cytology was also used as a diagnostic
tool for genital herpes.
Cellular changes caused by HSV can be recognized in
cervical scrapings after Papanicolaou stain, and in
lesion scrapings in Tzanck preparations.
However, using these changes to diagnose HSV is not
appropriate as they do not differentiate between HSV-
1and HSV-2, or between HSV and other viral
infections. Furthermore, the cytological techniques are
only 30%-80% as sensitive as cultures for HSV, and
have a low but significant false positive rate.
52
53. EHS
Belum jelas, ada kemungkinan :
- Infeksi primer akibat transmisi virus secara
langsung melalui jalur neuronal dari
perifer ke otak melalui saraf Trigeminus
atau Offactorius. Faktor precipitasi
adalah penurunan sistim imun host.
- Reaktivitas infeksi herpes virus laten dalam
otak.
- Pada neonatus penyebab terbanyak adalah
HSV-
HSV-2 yang merupakan infeksi
daapatan dari secret genital yang
terinfeksi pada saat persalinan.
53
54. EHS
Laboratorium :
• Analisis CSS : Pada minggu pertama dapat
normal, pleositosis mononuclear, peningkatan
ringan protein, kadar glucose normal/menurun
ringan, jumlah sel normal.
• Kultur CSS dapat dapat positif pada neonatus
• PCR : sensitive dan spesifik.
Radiologi : MRI : pilihan utama : lesi bermakna
pada lobus temporalis bagian medial dan
bagian inferior lobus frontalius.
EEG : cukup sensitive tapi tidak spesifik
Biopsi otak : pemeriksaan definitive untuk
menegakkan diagnosis
54
57. Recurrences and triggers
• Following active infection, herpes viruses
establish a latent infection in sensory and
autonomic ganglia of the nervous system.
The double-stranded DNA of the virus is
incorporated into the cell physiology by
infection of the nucleus of a nerve's cell
body. HSV latency is static—no virus is
produced—and is controlled by a number
of viral genes, including Latency
Associated Transcript (LAT).
57
58. Alzheimer's disease
• Scientists discovered a link between Herpes
Simplex Type I and Alzheimer’s disease in 1979.
In the presence of a certain gene variation
(APOE-epsilon4 allele carriers), HSV type 1
appears to be particularly damaging to the
nervous system and increases one’s risk of
developing Alzheimer’s disease. The virus
interacts with the components and receptors of
lipoproteins, which may lead to the development
of Alzheimer's disease. This research identifies
HSVs as the pathogen most clearly linked to the
establishment of Alzheimer’s.
• Without the presence of the gene allele, HSV type
1 does not appear to cause any neurological
damage and thus increase the risk of Alzheimer’s.
58
59. Bell's palsy
• A type of facial paralysis called Bell's palsy has
been linked to the presence and reactivation of
latent HSV-1 inside the sensory nerves of the
face (geniculate ganglia), particularly in a mouse
model. This is supported by findings that show
the presence of HSV-1 DNA in saliva at a higher
frequency in patients with Bell's palsy relative to
those without the condition.
• However, since HSV can also be detected in these
ganglia in large numbers of individuals that have
never experienced facial paralysis, and high titers
of antibodies for HSV are not found in HSV-
infected individuals with Bell's palsy relative to
those without, this theory has been
contested.[36] Other studies, which fail to detect
HSV-1 DNA in the cerebrospinal fluid of Bell's
palsy sufferers, also question whether HSV-1 is
the causative agent in this type of facial 59
paralysis.
63. Diagnosis
• Diagnosis is based on the physical examination
and patient history. Helpful (but nondiagnostic)
measures include laboratory data showing
increased antibody titers, smears of genital
lesions showing atypical cells, and cytologic
preparations (Tzanck test) that reveal giant cells.
• CONFIRMING DIAGNOSIS Diagnosis can be
confirmed by demonstration of the herpes
simplex virus in vesicular fluid, using tissue
culture techniques, or by antigen tests that
identify specific antigens.
63
64. Limitations of Type-Specific Serology
Type-
• Tests vary in their reliability and reproducibility.
• A positive test merely implies that the person has
been infected with one or both of these viruses at
some time in the past.
• Positive tests provide information about previous
exposure to one or both of these viruses, but do
not provide specific information about whether
particular genital symptoms are due to herpes.
• A positive test does not imply that the person is
infectious, although evidence suggests that the
majority of individuals who have antibodies to
one or the other of these viruses may shed the
virus asymptomatically or from unrecognised
lesions from time to time.
• Some patients appear to lose HSV-2 antibodies
with time using the current ELISAs
64
65. • PCR sensitivity rates vary from 75% to
100%
• The use of serology in the diagnosis of
neonatal HSV infection is hampered by
several factors. First, transplacental IgG
antibodies cannot be differentiated from
IgG produced by the infant. Second, the
ability of some severely affected infants to
make antibody is impaired. Third, the
commercially available assays for HSV IgM
antibodies have variable and limited
reliability.
65
66. Direct Testing
• Direct Testing (DFA and Viral Culture) is indicated
in active infections, but requires accurate
collection of specimens from lesions and so
cannot be used for asymptomatic genital herpes
infections.
• Specimens:
DFA: Collect infected cells from the base of the
vesicle or ulcer using an HSV DFA collection kit.
Viral Culture: collect infectec cells and vesicle
fluid using a viral transport swab
• Request: 'HSV DFA' and/or 'HSV culture'
Note: if DFA is positive, no further testing is
required; if DFA is negative and a viral transport
swab has been collected, viral culture will be
performed.
66
67. Serology
• Anti-HSV IgG is usually detectable 2-4 weeks
after primary infection. Specific IgM is detectable
in primary infections, but may also ocurr in
reactivation and so is not diagnostically helpful.
Serology is not recommended for the diagnosis of
acute HSV infections.
• However type specific HSV IgG serology for
immune status may be useful for:
• Epidemiology and couselling for 'in contact' or
'at-risk' patients.
• classification of HSV immune status in patients
with gential blisters or ulcers which are culture
negative
• prognosis (eg HSV-1 genital infectoin is not as
likely to recur as HSV-2 infection)
67
68. HSV Virology
• The HSV type 1 and 2 (HSV-1 or HSV-2) is a
large, double-stranded DNA virus with an
icosahedral nucleocapsid. The herpes virus
belongs to the Herpesviridae family, the
Alphaherpesvirinae subfamily, and the
Simplexvirus genera. The genomes of HSV-1 and
HSV-2 exhibit great homology, but the HSV-2
genome has an inherently higher mutation rate
than HSV-1. Important viral glycoproteins
include1: (1) gD, which is a potent inducer of
neutralizing antibody and is important in viral
attachment and entry into cells; (2) gB, which is
required for infectivity; (3) gH through gL, which
are important in viral attachment and entry into
cells; and (4) gG, which provides antigenic
specificity, allowing serologic differentiation of
HSV-1 from HSV-2 (gG-1 and gG-2,
respectively). 68
69. HSV Virology
• After initial infection, HSV virions spread
by retrograde axonal flow to sensory
ganglia, where the virions establish
latency.1 The neurovirulence of HSV is
attributable to the thymidine kinase
gene.1 Reactivation of the virus occurs
periodically in response to stressors (eg,
illness, fatigue, ultraviolet light, tissue
damage1) despite host humoral and
cellular immunity.
69
70. Several alternative cell types have been
suggested as origins of HSV excreted in
the oral cavity. Oral epithelium and, more
specifically, gingival sulcular epithelium
and ocular and salivary tissues have all
been proposed as sites of HSV replication.
Extra-oral persistence of HSV DNA in non-
neuronal tissues has been demonstrated
in skin, blood, and ocular tissue.
70