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Mercy CLS 1: Immunology > IFA + Hgb Electrophoresis > Flashcards

IFA + Hgb Electrophoresis Flashcards

1
Q

general principle of IFA

A

slide with substrate (HEP-2 or MSK) + pt sample with Ab + conjugate + fluorescent label

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2
Q

used in wash step and dilutions

A

PBS

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3
Q

fluorescent labels

A

FITC
rhodamine

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4
Q

darkens background

A

counterstain

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5
Q

major interference with IFA

A

rheumatoid factor (IgM anti-IgG)
an anti-IgM conjugate binds to RF and causes false pos
camouflages pt IgG, making it look like IgM

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6
Q

resolve RF interference

A

neutralize/adsorb RF or patient IgG when doing an anti-IgM assay

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7
Q

other interferences with IFA

A
  • light exposure — must be kept in dark when running, as fluorochrome is light-sensitive
  • insufficient washing — Ab not bound to slide may remain — false positives
  • drying out — need to be kept in a moist chamber while they sit — does not wash as well when dried out, false positives
  • reading of samples
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8
Q

reflex for any positive IFA

A

titer
serial dilution; most often twofold, but may begin with a higher dilution

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9
Q

QC requirement for IFA

A

positive and negative control on every slide you read (no matter how many wells on a slide)

may dilute out positive control to its endpoint (compare pt to the weakest positive)

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10
Q

sample requirements for IFA

A
  • usually use serum — red top
  • generally good for a few days in the refrigerator
  • generally good for weeks in the freezer, but no re-freezing (may go back in refrigerator)
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11
Q

HEP-2 substrate used for…

A

ANA

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12
Q

MSK substrate used for…

A
  • ASMA
  • APCA
  • AMA
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13
Q

Crithidia lucilliae substrate used for…

A

anti-nDNA

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14
Q

AMA fluorescence pattern

A

kidney tubules and parietal cells
cannot determine if an APCA is also present

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15
Q

C. lucilliae fluorescence pattern

A

kinetoplast (holds DNA); basal body may fluoresce, but not important

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16
Q

speckled + reflex

A

extractable nuclear antigen by EIA

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17
Q

4 ENA results and significance

A
  • Smith (Sm) —lupus
  • Ribonucleoprotein (RNP) —mixed connective tissue disease, lupus
  • SSA, SSB —Sjogren’s syndrome A and B
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18
Q

significance of anti-ssDNA

A

anti-ssDNA = anti-histones
seen in drug-induced lupus
d/c drug

19
Q

all of nucleus fluoresces
mitotic cells +

A

homogeneous
lupus, sometimes MCTD
reflex anti-nDNA

20
Q

spotted nuclei
mitotic cells =

A

speckled
reflex to ENA

21
Q

ladder-like appearance
mitotic cells +

A

centromere
CREST syndrome
no reflex

22
Q

large spots in nuclei
mitotic cells +

A

nucleolar
progressive systemic sclerosis

23
Q

outline of nuclei
mitotic cells +

A

peripheral/rim
highly specific for lupus

24
Q

spindle apparatus

A

spindle pattern
no clinical significance

25
Q

anti-nDNA seen in…

26
Q

disease associations for Abs tested for on MSK

A

ASMA – chronic active hepatitis
AMA – primary biliary cirrhosis
APCA – pernicious anemia

27
Q

Hgb is —- charged and migrates…

A

negatively
toward the +

28
Q

hgb electrophoresis interferences

A
  • application of sample — if you touch gel with pipette, it won’t be as smooth/even, and may add things to gel
  • gels can melt or dry out
  • gels must be stored flat
  • wrong voltage
  • pH of buffer and gel must be correct; can changes charges on proteins
29
Q

abnormal C/A2 band

A

reflex to A2 column chromatography

30
Q

explain A2 column chromatography

A
  • pt hemolysate passes through a positively charged resin
  • eluting buffer added to pull out hgb A2, which drips out into a tube
  • may not remove all of A2, particularly when hgb S is present
  • can be measured with spectrophotometry
31
Q

functions of reagents in Hgb electrophoresis:
buffer strips
stain
wash solution
destain solution

A

buffer strips — carries hgb through gel
stain — binds to hgb
wash solution — washes away extra stain
destain solution — clears background of stain

32
Q

hgb electrophoresis QC

A

every strip has to have an AFSC control; normal control not required but often performed

column chromatography requires a normal and abnormal control

33
Q

Hgb electrophoresis sample requirements

A
  • whole blood in EDTA
  • in refrigerator, minimally good for 3-4 days
  • should not be frozen
  • if an unstable hgb is suspected, run w/i 24 hours
34
Q

cellulose acetate gel

A

alkaline gel

35
Q

citrate agar

36
Q

alkaline gel migration pattern

A

++ A F S C =

with S: Hgb D, Hgb G, Hgb Lepore
with C: Hgb A2, Hgb E, Hgb O
Hgb H and Hgb Bart migrate before A (fast)

37
Q

4 beta chains, adults
4 gamma chains, babies

A

hgb H
hgb bart

38
Q

reflex with abnormal hgb electrophoresis

A

acid gel
possibly A2 chromatography
sickledex with Hgb S +

39
Q

acid gel migration pattern

A

++ C S A F =
Hgb A2 migrates with A

40
Q

normal adult hgbs

A

97% A
3% A2
<1% F

41
Q

no A, increased F and A2

A

beta thal major

42
Q

some A, increased A2

A

beta thal minor

43
Q

low A, increased F

Mercy CLS 1: Immunology (19 decks)

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