Laboratory #5 Fibrinogen

Laboratory #5 Fibrinogen Skills= 12 Points Objectives: The student should be able to:
1. 1Perform a fibrinogen on two (2) controls and one (1) patient within 2 SD of known values. 2. Properl report results with !"# accurac . $. Properl graph %ualit control results with 1&& # accurac . '. State the reference range for fibrinogen. ". (nterpret the results of fibrinogen testing. ). *+plain the clinical significance of the ,ibrinogen test.

Materials:
1. 2. $. '. ". ). -. 9. !. 1&. 11. 112 + -" test tubes and rack Pipettes and tips. 1&& /0 and 2&&/0 1+ford and a 1.& m0 serological in increments of &.1 m0 Pipette tips 2imwipes or gau3e 4imer 5alcium thrombin reagent 1wren6s 7eronal 8uffer 5lotting 4ime (sec.) vs. ,ibrinogen (mg:d0) data table 5ontrols. 0evel ((normal) and 0evel $(abnormal) Patient citrated plasma specimen (nstrument (25;1Delta or 5ascade <;')

References: 1. 2. $. '. ". 1Powers= 0awrence >.= Diagnostic ?ematolog = pp. '9"= '!&. 8rown= 8. @.= ?ematolog . Principles and Procedures= "th edition= p. 21!. 4urgeon= <ar 0ouise= 5linical ?ematolog 4heor and Procedures= ,irst edition= pp. '&';'&) 25 1 Delta package insert= Sigma Diagnostics. ?elena 0aboratories 5ascade <;' 1peratorAs <anual

Clauss Principle for Fibrinogen Quantification:

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MLAB 1227-Fibrinogen

Laboratory #5 Fibrinogen

4he basic principle of the 5lauss ,ibrinogen test is high levels of thrombin are added to diluted plasma and the time it takes for that thrombin to convert fibrinogen to fibrin correlates to the concentration of fibrinogen when compared to a standard curve. 1,ibrinogen (() thrombin( ((a) B ,ibrin clot ((a)

4he plasma is diluted (usuall 1.1& but this ma var if the fibrinogen concentration is ver low or ver high) to minimi3e the effect of Cinhibitor substancesD within the plasma (heparin= elevated levels of ,DPs.) 4he use of a high concentration of thrombin ensures that the clotting times are independent of thrombin concentration over a wide range of fibrinogen levels. @ calibration curve is constructed using reference plasma (calibrator) of known fibrinogen concentration b preparing a series of dilutions (1." E1.'&) in buffer to give a range of fibrinogen concentrations. 4he clotting time of each of these dilutions is established (using duplicate samples) and the results are plotted on graph paper concentrations in mg:d0 on the +;a+is= versus the clotting time in seconds on the ;a+is. 4here should be a linear correlation between clotting times in the region of 1&;'& seconds. 4he test specimen (platelet;poor plasma) is also diluted 1.1& in buffer. 4hrombin reagent is added. 4he time taken for the clot to form is compared to a calibration curve and the fibrinogen concentration in mg:d0 is deduced. 4he clotting times are inversel proportional to the concentration of fibrinogen in each sample= e.g.= a short clot time corresponds to a high concentration of fibrinogen.

Fibrinogen Reference range: 2&&;'&& mg:d0

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Laboratory #5 Fibrinogen

Clinical Significance: 14here are several causes for a deficienc of fibrinogen. Severe hemorrhaging ma result in an case. @fibrinogenemia (a lack of fibrinogen) or a d sfibrinogenemia (abnormal fibrinogen) ma be congenital. @c%uired deficiencies ma be due to liver disease= disseminated intravascular coagulation (D(5)= or during a therapeutic plasma pheresis procedure. *levated fibrinogen levels ma be found in pregnanc = following surger = or in patients in a h percoagulable state such as with thrombosis. NOTES: 4he linearit range for this lab will be the range between ,ibrinogen level associated with 1." dilution and the fibrinogen level of the 1.'& dilution 1,or fibrinogen values out of the linearit range a 1.1& dilution of the plasma will not work and a different dilution must be used. ,or e+tremel high fibrinogen levels= a 1.2& dilution of the plasma is used for the procedure. ?owever= due to the change in dilution= the result read off of the fibrinogen data table must be multiplied b a factor of 2 (since our 1.2& dilution is 2 times the 1.1& dilution). ,or e+tremel low fibrinogen levels (F') mg:d0) a 1." dilution of the plasma is used for the procedure. 4he result read off of the data table must then be divided b a factor of 2 4he ,ibrinogen test is performed at $-G5= but the 4hrombin is maintained at room temperature. Duplicate clotting time results must be it!in " #$% seconds of eac! ot!er or t!e test s!ould be repeated in duplicate again$

Qualit& Control: Hualit control materials (normal:0evel 1 and abnormal:0evel $) with established control limits should be run. I550S recommends controls are tested at the beginning of each testing da = followed b testing during each subse%uent shift or with each batch of assa s. @dditionall = %ualit controls should be performed with an part or reagent change as well as noticeable shifts or trends in the H5 data. @dherence to good laborator practice and careful following of the recommended procedures will result in clinicall accurate and reproducible results.
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Laboratory #5 Fibrinogen

1nce reagents and controls are reconstituted= put a time= toda As date= e+piration date and technologist initial on the bottle.

Cascade '() Standard Curve Procedure: 1. @llow all reagents to e%uilibrate to room temperature. 2. Prepare 1."= 1.1&= 1.2&= 1.$& and 1.'& dilutions of ,ibrinogen 5alibrator using 1wrenAs 7eronal 8uffer as follows.

Reagent
8uffer in m0 5alibrator in m0

*:%
&.9 &.2

*:*#
&.! &.1

*:+#
1.! &.1

*:,#
2.! &.1

*:)#
$.! &.1

$. Jun duplicate determinations on each dilution of the fibrinogen calibrator as follows. a. ,rom original screen. Press FStdB b. Jun Standard. Press F*nterB c. Select test t pe. Press F,ibB then F*I4*JB d. Delete old standardK Io Les Select Les b pressing the FMB ke *nter user (D N O ((f prompted) using the number ke s and press F*I4*JB e. *nter 0ot Iumber using number ke s and press F*nterB f. *nter Jeference 7alue from ,ibrinogen 2it for undiluted calibrator b pressing FDelB and removing old value = using number ke s= and press F*I4*JB Select dilution. 1." Press F*I4*JB PPJepeat for each dilution and press FMB to select dilutions for 1.1&= 1.2&= 1.$&= and 1.'&PP g. Jun specimens in duplicate for each calibrator dilution as outlined in the 5ascade <;' 4est procedure steps 2i;2n found below. h. 5ontinue to run dilutionK Io NLesO Press F*nterB i. Plot the clotting times on the ,ibrinogen Standard 5urve found below. Cascade '() Test Procedure: 1. Dilute patient plasma and controls 1.1& with 1wrenAs 7eronal 8uffer (1 part specimen and ! parts buffer) 2. 4est patient and control dilutions in duplicate in the following manner. a. 4urn on instrument and allow s stem to initiali3e.
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Laboratory #5 Fibrinogen

b. 2eep 4hrombin Jeagent at room temperature c. 4he screen will read C4est <enu. Select. Jun 4ests MD Press F*I4*JB d. 4he instrument is set to run each sample in duplicate. 4he ne+t screen will read CDuplicate all same patient (DKD Select I1 and press F*I4*JB e. Select channels to be run b pressing F1B for channels 1 and 2 or press F$B for channels $ and ' to be run in duplicate. 4hen press F*I4*JB f. *nter Patient (D or control (D g. Select ,(8 as test t pe h. Put cuvette in optics chamber and close lid i. Pipette 2&& Q0 of diluted specimens into the cuvette and press lid (beep sounds) R. Discard tip k. <i+ 4hrombin reagent l. >hen prompted= add 1&& Q0 of 4hrombin Jeagent= press lid (beep sounds) m. Discard tip n. Jemove cuvette(s) and press C*nterD for ne+t test. -C(* Standard Curve Procedure: 1. Prepare dilutions as described in the 5ascade <;' standard curve procedure. Jefer to chart. 2. 5ontinue to the 25;1 procedure below= starting at step '. *ach dilution of the fibrinogen calibrator should be ran in duplicate. $. 1nce all fibrinogen calibrators have been run= calculate the mean time for each reference dilution. Plot the calibration curve on graph paper. 4his curve is used to interpret the fibrinogen results for the controls and patient samples. -C(* Test Procedure: 1. @llow thrombin to warm to room temperature= at least 1" minutes. 2. Prepare controls and patient plasma b making a 1.1& dilution. Place 1&& Q0 of control to !&& Q0 of 1wrenAs 8uffer into a plastic tube. <i+ gentl . Store refrigerated until read to use. $. Place cuvettes in incubation wells. '. Pipette 2&& Ql diluted sample into the bottom of each cuvette. Sample should be pipette into the bottom of the cuvette opposite the ball without touching either side or the bottom of the cuvette. ". Discard tip. ). Select ,(8 as the measuring mode (touch button F2B=then F'B) -. 4he incubation time starts to count down. 4he incubation time is set for )&;19& seconds. a. (f the incubation needs to be changed= the technologist can adRust the incubation time within ' seconds after countdown begins. (Set to new incubation time using (F$BSF2B). 4o re;start incubation time use (F2B).
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Laboratory #5 Fibrinogen

9. 5losel spaced warning beeps will begin when there is 2& seconds incubation time remaining and will continue at ";second intervals until the incubation time is complete. Tust prior to the end of the incubation= aspirate 1&& Q0 of thrombin reagent into pipette tip. >hen incubation ends and ST.RT is displa ed= press F$B. >ith the P:< in the displa = dispense 1&& Ql thrombin reagent into the cuvette= towards the right of the ball. Simultaneousl press F'B. a. (f a C4(<*1U4D appears in the displa = timer should be reset. 4o reset the timer= press and hold button F'B. Press button F2B. !. 4he timer stops automaticall when clotting begins and the ball is pulled from the stead position. Jecord results= including measurement units of seconds. 1&. Jemove cuvette from the test well and discard into a bioha3ard container. Position the ne+t sample containing cuvette in the test well. 11. Press button F2B to proceed to the ne+t sample. Jepeat steps ";11. 4he averaged result is then used to read the actual fibrinogen concentration in mg:d0 off the fibrinogen data table. (t is this value that is reported out as the final result.

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MLAB 1227-Fibrinogen

Laboratory #5 Fibrinogen F B! "#$%" &TA"'A!' ()!*%

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MLAB 1227-Fibrinogen

Laboratory #5 Fibrinogen

Student Iame.VVVVVVVVVVVVVVV /ab 0 %: Fibrinogen

*+ pts$

Standard Curve Results

Fibrinogen Dilution
*:% *:*# *:+# *:,# *:)#

Run *

Run +

.verage

Concentration

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Laboratory #5 Fibrinogen

&tudent "a+e:,,,,,,,,,,,,,,,,,, Control and Patient Results ##$%art &'ality $ontrol res'lts on gra(% ## )ncl'*e +eas're+ent 'nits
Fibrinoge n ,or+al control 2bnor+al control Patient ,a+e-).# /'n 1 /'n 2 /'n 3 $lotting 0i+e 12vg3 Fibrinogen $oncentratio n 4it%in /e5erence /ange6

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Laboratory #5 Fibrinogen

,a+e8888888888888888888 .ate88888888888888888888 Lab #5: Fibrinogen St'*y &'estions Points= 14

1- 11>hat dilution of plasma is usuall used for fibrinogensK(1pt)

a. ?ow is this dilution preparedK (1pt)

b. >hat diluent is usedK (1pt)

.-

>hat should be done if the patient6s fibrinogen level is above linearit for the standard curve ou are usingK (1pt)

d-

>hat should be done if the patient6s fibrinogen level is below linearit for the standard curve ou are usingK (1pt)

e. (s the control dilutedK (1pt)

2-

1(f the fibrinogen procedure ou perform results in a large number of seconds for the clot to form= what do ou e+pect the actual fibrinogen concentration in mg:d0 to be= high or lowK (1 pt)

$. >hat reagent is added to the diluted plasma in the fibrinogen procedureK (1 pt)

'. >hat is the reference range for fibrinogenK (1 pt)

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Laboratory #5 Fibrinogen

". 0ist at least three ($) causes of a decreased fibrinogen. (1." pts)

/- 10ist at least three ($) causes for an increased fibrinogen.(1." pts)

-.

@t what temperature are fibrinogens performedK(&." pts)

0- ?ow closel should duplicate results agree in order to be acceptableK(&." pts)

1- 1Using the fibrinogen data table created during our lab procedure= what is the final fibrinogen concentration of a patient with the following results(1 pt)

5lotting times.

1&.$ sec 11.- sec 1&.1 sec

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MLAB 1227-Fibrinogen