Coag lab exam Flashcards
2 platelet count methods
- manual (phase)
- automated analyzer
platelet count sample
EDTA WB
platelets are manually counted with the scope in ——– and on —x
phase
40
diluent for plt count and function
1% ammonium oxalate
- preserves plts, WBCs, nRBCs
- lyses RBCs
plt count dilution
1:100
1.98 mL ammonium oxalate + 20 μl blood
a plt count dilution is good for —– hours
3
plt clumping or satellitism
cause
solution
EDTA sensitivity
recollect in Na citrate
3 methods of automated plt ct
- impedance (PLT-I)
- optical (PLT-O)
- fluorescent (PLT-F)
electric current is blocked as plts pass through
based on size only
impedance method
impedance method of plt count interferences
- giant plts
- schistocytes
- small microcytes
- plt clumps
uses light scattering properties to distinguish plts based on internal complexity
optical method
uses fluorescent dye to stain plt organelles
fluorescent method
RR and critical range for plt count
150-400 x 10^3/μL
<20 x 10^3/μL
performed when plts are “flagged” on analyzer to verify accuracy of instrument’s count
platelet estimate
uses a 20 μL capillary tube for dilution
manual platelet count
let plt dilution sit for —– mins to allow RBCs to lyse
5
which squares are plts counted in?
5 smaller squares (area of 0.2 mm^2)
if plt count <40 in these squares, count entire large center square
plt count calculation
plts/μL = (plts counted)(100)(10)/0.2
100 = DF
0.2 = area
plt estimate procedure
- scan entire feathered edge on 10x for clumps/fibrin strands
- in monolayer, count platelets in 5 fields and find average
- multiply average by 15 and 20 to get a range
tests associated with primary hemostasis
- plt count
- bleeding time
- plt function assay
- plt aggregation studies
- flow cytometry
tests associated with secondary hemostasis
- PT
- PTT
- TT
- Fibrinogen
tests associated with fibrinolytic system
- FDPs
- D-dimers
things to avoid during venipuncture
- TF entering sample
- do not draw tube with anticoag or clot promoter before the Na citrate
- hemolysis
- heparin contamination
how to avoid heparin contamination
- flush line with saline
- discard 1st 5 mL blood
coag tubes are —– Na citrate with —— anticoagulant:blood ratio
3.2%
1:9
function of Na citrate
chelates Ca++ in blood
if there is a sample volume <85% with Na citrate…
test results must be prolonged because excess additive will chelate Ca++ in the reagents
Hct —— may prolong test results because less plasma requires less Na citrate
> 55
what makes Na citrate a good additive?
- good preservative for factor V
- sensitive to heparin, so pts can be monitored
- better for ABS reading instruments because it doesn’t create precipitate
——– plasma is used for most coag tests
procedure
platelet poor
spin to reach plt <10,000/μL
handling and longevity of plt poor plasma
- good 4 hours RT or refrigerated
- 2 hours if pt on heparin
- keep capped until analyzed
- no repeated freezing/thawing
——— plasma is used for plt aggregation studies
procedure
platelet rich
spin at lower speed to obtain plt count of 200-300 x 10^3
handling and longevity of plt rich plasma
- good for 3 hours RT
- do not freeze
why can’t glass be used for coag samples?
negative charge causes contact activation of intrinsic pathway
old screen for platelet function
measures primary plt plug
bleeding time
factors that affect bleeding time
- plt function
- plt count (<100 will prolong, do count first)
- vessel integrity
- aspirin, clopidogrel (avoid 7 days prior)
- technique
automated screening method for plt function, which replaced bleeding time
platelet function assay
PFA uses a cartridge containing…
a collagen membrane coated with platelet agonist, such as epinephrine or ADP
collagen/epinephrine membrane used in PFA for…
plt dysfunction due to plt defects or inhibitors
collagen/ADP membrane used in PFA to…
determine if abnormal epinephrine result is due to aspirin
aspirin effect on PFA
epinephrine abnormal, ADP normal
what affects bleeding time but not PFA?
vessel integrity
sample handling for PFA
- do not spin
- cannot go through pneumatic tubes
- must stay at RT
- run within 3 hours
aggregating agents added
light transmission increases
aggregation studies
used to measure light transmittance for plt aggregation
aggregometer
aggregating agents
- collagen
- ADP
- epinephrine
- ristocetin
- arachidonic acid
no ——– 7 days prior to plt aggregation study
aspirin
monophasic pattern of plt aggregation
collagen
ristocetin
arachidonic acid
biphasic pattern of plt aggregation
ADP
epinephrine
biphasic pattern of plt aggregation occurs when…
ADP released from dense bodies of plts
ristocetin represents ———– of plts and depends on interaction of —- and —–
agglutination
vWF and GPIb
how to tell difference between ristocetin aggregation from VWD and from Bernard-Soulier syndrome
VWD: corrects with normal plasma added
BSS: does not correct with normal plasma added
flow can be used to dx…
Bernard-Soulier syndrome
Glanzmann thrombasthenia
most reagents for 2°hemostasis tests are ———– and must be reconstituted with ———-
lyophilized
purified water (NERL)
when does QC for the 2° hemostasis tests need to be done?
- every 8 hours
- new reagent bottle
- after maintenance
- after problem with instruments or reagnets
fibrin endpoint systems detect…
unstable fibrin clot, factor XIII not yet activated (so XIII problems not detected)
2 automated methods for 2° hemostasis tests
- optical (ABS reading)
- mechanical (use a magnetic bead)
STAGO
evolution and compact instruments
use magnetic bead for 2° hemostasis tests
mechanical 2° hemostasis methods are not sensitive to…
hemolysis, icerus, lipemia
POC test used to estimate effectiveness of protamine sulfate dose (used to absorb heparin after cardiac surgery)
whole blood clotting time
—- used to monitor coumadin tx
—- used to monitor heparin tx
PT
PTT
not sensitive to slightly ↓ fibrinogen (must be <100 mg/dL)
PT
uses TF preparation to activate cascade by forming TF/VII complex and detecting clot
PT
PT RR
12-15 sec
how does each lab establish its PT RR?
run at least 30 normal plasma samples, half male and half female
——— will not interfere with PT unless ↑↑
heparin
absorbent in reagent
coumadin action
inhibit production of vitamin K dependent factors in liver
used to standardize PT results for coumadin monitoring
international normalized ratio
ISI
international sensitivity index
supplied by manufacturer of each lot of thromboplastin for PT
INR =
INR = R^ISI
R = PR ratio = pt PT/mean normal PT
ISI supplied with reagent
INR TR
2.0-3.0
factors that affect efficacy of oral anticoag
- dosage
- vitamin K in diet
- body mass
- drugs (antibiotics, aspirin)
- liver function
why do antibiotics affect coumadin dosing?
↓ normal flora
↓ vitamin K made by them
↓ dose
↑ INR at risk for…
bleeding
3 uses for PTT
- detect deficiencies in intrinsic/common pathway
- monitoring heparin therapy
- detecting inhibitors
activated partial thromboplastin provides PL surface and activator with negative surface
CaCl2 provides Ca++
PTT
activators present in PTT reagent
kaolin
silica
PTT RR
25-35 sec
coag factors must be ——% of normal or less to cause abnormal PTT
25-40
is sensitive to slightly ↓ fibrinogen
PTT
better than PTT for monitoring heparin therapy
anti-Xa test
function of heparin
enhances action of ATIII on thrombin and Xa
neutralizes IXa and XIa
run PTT —- hours after changing heparin dose
6
there is no standard, such as INR, available for…
PTT monitoring of heparin
how is heparin therapeutic range determined?
correlating PTT results from patients on heparin with Xa inhibition results
neutralize heparin
- PF4 (trauma, cold storage)
- protamine sulfate
Xa inhibition assay should be used…
- acute phase reactants present (early in DVT, PE)
- LMW heparin used
action of acute phase reactants on heparin
compete with ATIII for heparin binding sites, making PTT unreliable
used anti-Xa assay
prophylactic heparin given IM
usually doesn’t need monitoring
LMW heparin
advantage of LMW heparin over unfractionated heparin
unfractionated heparin patients tend to make platelet Ab
LMW heparin does not cause this
anti-Xa is a ———– assay using a standard curve
chromogenic
factor Xa inhibition assay TR
0.3-0.7 U/mL
deheparinzed plasma reagent can absorb up to —— heparin
10 mL
use of deheparinized plasma
- remove heparin contamination
- for coumadin effect when pt is being converted from heparin to coumadin (if PT reagent sensitive to heparin)
mesaures conversion of fibrinogen to fibrin
TT
function of TT
look for circulating thrombin inhibitors
rate limiting factor in TT is…
thrombin in pt plasma
TT RR
15-19 sec
5 causes of abnormal TT
- FDPs
- ↓ fibrinogen
- dysfibrinogenemia
- thrombin inhibitor
- heparin
major difference between TT and Fib
TT: low level thrombin + undilute plasma
Fib: excess thrombin + dilute plasma
used to quantitate fibrinogen
Fib
Clauss assay reference method
Fib
TT performed on 1:10 dilution of plasma and control
Clauss method of Fib
rate limiting factor in Fib is…
pt fibrinogen concentration
Fib is ———– to TT
inversely proportional
Fib RR
150-350 md/dL
performed if PT and PTT are normal, but pt is bleeding or clotting
FDPs/D-Dimer
FDP in plasma indicates —– fibrinolytic activity
↑
FDP method
serum latex agglutination
conditions that can cause ↑ FDPs
DIC
liver/kidney/heart disease
MI
carcinoma
PE
DVT
eclampsia
D-dimer has excellent ———- predictive value for DVT
negative
4 parts of DIC panel
PT
PTT
fibrinogen
D-dimer
↑ FDPs
normal D-dimer
fibrinogenolysis
differentiates factor deficiencies from circulating inhibitors
mixing studies
mixing study corrected =
mixing study not corrected =
factor deficiency
inhibitor
2 rounds of mixing studies
immediately
after 2 hours 37° incubation
time/temp dependent inhibitor
VIII
abnormal mixing study is followed by…
factor or inhibitor assay
uses single factor deficient substrate to determine if dilutions of pt plasma correct it
factor assay
factor assay corrected =
patient is not deficient
used with TT to differentiate FDPs, dysfibrinogenemia, and heparin contamination
reptilase time
enzyme that cleaves fibrinopeptide A
reptilase
reptilase time RR
18-22 sec
———- time not affected by heparin
reptilase
TT ↑↑
reptilase ↑
FDPs
TT ↑
reptilase ↑↑
dysfibrinogenemia
TT ↑↑
reptilase normal
heparin contamination
uses extended incubation with first reagent in PTT
prekallikrein screening test
PK screen corrects =
prekallikrein deficiency
used to detect XIII deficiency
urea solubility test
fibrin clot is insoluble in 5M urea if…
XIII is present
5M urea clot dissolves =
XIII deficiency
plasma clotted with CaCl2
5M urea added
24 hour RT incubation
XIII screening test
XIII must be ——% to be detected and to be clinically significant
<1-2%
2 general types of assays for factor deficiencies
- activity assays - determine function
- immunoassays - determine quantity
RCoF assay
vWF activity assay
measures ability of vWF to agglutinate standard suspension of platelets in presence of ristocetin
RCoF assay
microtiter ——– used for vWF:Ag immunoassay
ELISA
↓ vWF:Ag
type I and III VWD
normal vWF:Ag
type II VWD
used to confirm type of VWD
SDS PAGE multimer analysis
all multimers of vWF ↓
VWD I
only HMW multimers of vWF ↓
VWD II
all multimers of vWF absent
VWD III
2 most common factor inhibitors
- lupus-like anticoagulant
- VIII inhibitor
inhibitor causing clotting
lupus-like
anti-PL inhibitor
lupus-like
3 steps to ID lupus-like inhibitor
- clotting assay (prolonged PTT)
- mixing studies
- reduce or add excess PL to see if it corrects
plt neutralization procedure for LLAC
- ruptured platelets neutralize anti-PL
- if PTT corrects, LLAC is present
false pos with heparin; PF4 neutralizes it
LLAC methods that use ↑ PL and ↓ PL
↑ PL: platelet neutralization; STAclot LA
↓ PL: dRVVT
dRVVT
dilute russel viper venom test
LLAC activity ↑ as PL content ↓
dRVV activates —– in plasma
X
abnormal dRVVT ratio =
LLAC present
uses PTT reagent with increased PL content
STAclot LA
EIA test to pick up configurations of anti-PL that other tests don’t detect
anti-cardiolipin Ab test
LLAC can bind PL on ——–, blocking ——–
VECs
protein C
uses dilute pt plasma with normal pooled plasma that has a known VIII activity
factor VIII inhibitor assay
thrombophilia
hypercoagulable state
repeated thrombotic episodes
ATIII assays use ——- as a target
thrombin or Xa
during ATIII chromogenic assay, thrombin releases ————- from substrate
p-nitroaniline
more ATII = —— color
↓
inherited deficiencies of ATIII
- ↓ ATIII
- dysfunctional ATIII
acquired deficiencies of ATIII
- DIC
- liver disease
- nephrotic syndrome
- OC/estrogen
- malignancy
protein C is quantitated by —– assay
EIA
protein C assay uses ——– to activate PC
thrombin or thrombomodulin complex
protein C assay is based on ability of PC to ———— PTT by…
prolong
inactivating Va and VIIIa
acquired protein C deficiencies
- DIC
- vit K deficiency
- liver disease
- oral anticoag
- postsurgical
2 forms of protein S
- free (40%), serves as cofactor
- bound to C4b (60%)
acquired protein S deficiencies
- liver disease
- pregnancy
- OC
- DIC
- T1DM
- oral anticoag
deficiency of —— or excess of ——- seen in in inflammatory states, associated with hypercoagulation
t-PA
PAI-1
useful in monitoring fibrinolytic tx (used to dissolve existing clots)
antiplasmin assay
used to detect factor V leiden mutation
activated protein C resistance test
factor V leiden mutation causes…
V to be resistant to degradation by protein C
—- should be initially normal or FVL mutation test is invalid
PTT
used to confirm FVL mutation dx
PCR
effect of prothrombin mutation
↑ prothrombin levels up to 30% higher than normal
risk for thrombosis
deficiency of enzyme needed to convert homocysteine to methionine
methylene tetrahydrofolate reductase mutation
useful for TTP dx
ADAMTS-13 mutation or auto-Ab
vWF-cleaving protease
ADAMTS-13
unusually large vWF multimers
TTP
associated with ADAMTS-13 mutation
associated with auto-Ab against enzyme
chronic relapsing TTP
acute TTP
—— assay used for ADAMTS-13 antigen or Ab
ELISA
