LIKING LIPAEMIARENÉE TIETJENS

WSCL HAEMATOLOGY

HAEMOGLOBIN

• A PROTEIN MOLECULE FOUND IN RED BLOOD CELLS.

• DELIVERS OXYGEN TO THE BODY’S TISSUES AND REMOVES

CARBON DIOXIDE VIA THE LUNGS.

• 2 ALPHA AND 2 BETA GLOBIN CHAINS EACH CONTAINING A

HAEM GROUP THAT CONTAINS AN IRON MOLECULE.

• REQUESTED BY A CLINICIAN AS PART OF A FULL BLOOD

COUNT AND IS USED IN THE INVESTIGATION OF ANAEMIA.

SYSMEX XN-1000• THE SYSMEX XN-1000 ANALYSER USES THE SLS-HB METHOD WHICH MEASURES HB USING

LIGHT ABSORBANCE.

• ALL CELLS ARE LYSED BY REAGENT.

• HYDROPHOBIC PORTION OF SLS CAUSES A

CONFORMATIONAL CHANGE TO EXPOSE THE HAEM

UNIT.

• FE2+ IS OXIDISED TO FE3+.

• HYDROPHILIC GROUP OF SLS BINDS FE3+ TO

FORM A STABLE COLOURED PRODUCT.

• THIS SLS-HB IS MEASURED PHOTOMETRICALLY.

• LIGHT ABSORBANCE IS PROPORTIONAL TO HB

CONCENTRATION.

LIPAEMIA

• CAUSES TURBIDITY IN THE LYSATE.

• FALSELY ELEVATES THE ABSORBANCE OVERESTIMATION OF HB

CONCENTRATION

• THE HB IS USED TO CALCULATE THE MCH (HB/RBC) AND MCHC (HB/HCT)

• RESULTS NEED TO BE CORRECTED.

PLASMA HB• DETECTION:

• FALSELY INCREASED HB RESULTS IN AN ELEVATED MCHC:

MCHC= HAEMOGLOBIN____

HAEMATOCRIT

•

MCHC >365 AND

Difference between the RBC-O and RBC-I is

<0.15

IT3000 Algorhyt

hm

“?Lipaemia” Flag

PLASMA HB• CORRECTION:

• PORTION OF SAMPLE IS CENTRIFUGED AND PLASMA SEPARATED INTO ANOTHER

TUBE

• PLASMA ANALYZED TO GIVE A PLASMA HB VALUE

• THIS IS USED TO CALCULATE A MORE ACCURATE HB VALUE.

• CORRECTED HB = HB – ((PLASMA HB X (1-HAEMATOCRIT))

• THE CORRECTED HB IS THEN USED TO RECALCULATE THE MCH AND MCHC.

• INEFFICIENT, LABOUR INTENSIVE AND LEAVES A LOT OF ROOM FOR ERROR.

• ~1 SAMPLE PER WEEK

• ~15- 20 MINUTES PER SAMPLE

WHY WE IT

1. DELAYS THE TRUE RESULT BEING SENT TO THE CLINICIAN, WHICH CAN DELAY TREATMENT.

2. THIS CAN ALSO DELAY OTHER SAMPLES FROM BEING ANALYSED AS THE ANALYSER IS TAKEN

OFFLINE TO RUN THE PLASMA MANUALLY (I.E. NO OTHER SAMPLES CAN RUN THROUGH)

3. OPENING A SAMPLE TUBE IS A HEALTH AND SAFETY RISK TO STAFF.

4. IF PIPETTING IS NOT DONE CAREFULLY, RED CELLS CAN BE TRANSFERRED ALONG WITH THE

PLASMA INTO THE NEW TUBE FOR REANALYZING. THIS WILL FALSELY INCREASE THE PLASMA

HB RESULT LEADING TO A FALSELY LOWERED FINAL HB.

5. A LARGE AMOUNT OF SAMPLE IS USED

a) INSUFFICIENT SAMPLE FOR FURTHER TESTS

b) CANNOT BE DONE ON PAEDIATRIC SAMPLES

COULD THERE BE A BETTER WAY?

OPTICAL HB (HB-O)

• RESEARCH PARAMETER ON THE XN20 ANALYSER.

• ANALYSED IN THE RET CHANNEL (I.E. THE PART OF

THE MACHINE THAT MEASURES THE

RETICULOCYTE COUNT)

• ANALYZES THE RED CELLS WHOLE, SINGLY AND

USING FLOW CYTOMETRY.

• CALCULATED FROM THE RBC-O AND RBC-HE (MCH

EQUIVALENT):

HB-O= RBC-O X RBC-HE

WHY WE IT1. NO INTERFERENCE BY LIPAEMIA AS CELLS ARE MEASURED INSTEAD OF A LYSATE.

2. SIMPLER METHOD WITH IMMEDIATE RESULT

3. CLOSED SYSTEM

4. THE CLINICIAN WOULD RECEIVE THE TRUE HB RESULT WITHIN A SHORTER TIME

FRAME

5. NO IMPACT ON THE TURNAROUND TIME FOR OTHER PATIENT SAMPLES

6. NO EXTRA SAMPLE AMOUNT IS REQUIRED (UNLESS THE RET CHANNEL WASN’T

UTILIZED ON FIRST RUN I.E. FOR RETICS)

HOW DOES IT COMPARE WITH THE PLASMA HB METHOD?. . . .

METHOD

• 31 SAMPLES WHICH FLAGGED FOR LIPAEMIA WERE CORRECTED USING THE PLASMA HB METHOD

• SOME SAMPLES WERE ANALYSED IN THE RET CHANNEL INITIALLY. I.E. RUN FOR RETICULOCYTES

• IF NOT, THE SAMPLES WERE RETRIEVED AND REANALYSED IN THE RET CHANNEL TO GIVE THE HB-O RESULT

• THE MCH, MCHC AND THEIR OPTICAL EQUIVALENTS (RBC-HE AND MCHC-O) WERE ALSO NOTED

• “NORMAL” SAMPLES (LIPAEMIA INDEX <10, MCHC <365) WERE ALSO OBTAINED TO COMPARE THE HB-O WITH THE REPORTED HB AND PROVE THAT THESE CAN BE INTERCHANGEABLE

RESULTSLIPAEMIC SAMPLES

• NUMBER OF SAMPLES: 31

• HB RANGED FROM:60-183 G/L

• LIPAEMIA INDEX RANGED FROM :62-3694

• % DIFFERENCE IN HB:0.0-6.9% (OUTLIER OF 16.9%)

• MEAN % DIFFERENCE:3.4%

NON-LIPAEMIC SAMPLES

• NUMBER OF SAMPLES: 31

• HB RANGED FROM:81-162 G/L

• LIPAEMIA INDEX RANGED FROM :1-10

• % DIFFERENCE IN HB:0.0-5.0%

• MEAN % DIFFERENCE:1.8%

MOU= 3.9%

OUTLIER• 14.03.2017

• HB=135

• CORRECTED HB=122 VS HB-O=103 (% DIFFERENCE 16.9%)

• HAEMOLYSIS 115, LIPAEMIA 1294 – WITHIN RANGE OF THE OTHER SAMPLES.

• CLINICALS – RENAL PATIENT

• 13.03.2017• CORRECTED HB= 103

• 15.03.2017• CORRECTED HB= 102

HUMAN ERROR WITH PLASMA HB METHOD?

RESULTS

y = 0.9804x + 1.4366R² = 0.9792

0

20

40

60

80

100

120

140

160

180

200

0 20 40 60 80 100 120 140 160 180 200

Hb

-O

Corrected Hb

Lipaemic Hb Comparisony = 1.0538x - 6.1806

R² = 0.9858

0

20

40

60

80

100

120

140

160

180

0 50 100 150 200

Hb

-O

Reported Hb

Non-Lipaemic Hb Comparison

THE HB-O CAN BE USED WHEN A SAMPLE IS LIPAEMIC,

INSTEAD OF USING THE PLASMA HB METHOD TO

CORRECT THE ORIGINAL HB RESULT.

TO NOTE - HAEMOLYSIS

• FLOW CYTOMETRY REQUIRES WHOLE CELLS TO ANALYSE.

• IN VITRO HAEMOLYSIS CAN BE CAUSED BY SOME PRE-ANALYTICAL

PROBLEMS SUCH AS DELAY, ROUGH TRANSPORT OR THERMAL IMPACT.

• THESE WILL LEAD TO RED CELL DESTRUCTION PROVIDING AN

UNDERESTIMATION OF HB-O.

• THE SLS-HB WITH PLASMA HB CORRECTION METHOD WILL NEED TO BE USED

IN SUCH CIRCUMSTANCES AS IT USES ABSORBANCE OF FREE HAEMOGLOBIN.

TO NOTE - HIGH WCC

• HB FALSELY ELEVATED WITH MARKEDLY HIGH WCC - LEADING TO AN

INCREASED MCHC AND MCH

• WE CANNOT REMOVE WHITE CELLS FROM A SAMPLE AND RUN IT AGAIN TO SEE

IF IT GIVES A RESULT THE SAME AS THE HB-O FROM THE ORIGINAL RUN

• NO WAY OF TESTING WHETHER THE HB-O IS ACCURATE OR NOT SO IT CANT BE

VALIDATED

TO NOTE - QUALITY CONTROL

• THE HB-O PARAMETER IS NOT MONITORED BY ANY EXTERNAL QUALITY

CONTROL AND IS NOT INCLUDED IN THE LIST OF TESTS ON SNCS.

PROPOSAL . . .

• THE HB-O RESULT CAN BE SENT THROUGH TO IT3K WITH THE REST OF THE FBC RESULTS WHEN THE "?LIPAEMIC" FLAG IS GENERATED

• ?"LIPAEMIA" IS A DROP DOWN BOX SO WHEN CLICKED ON CAN HAVE THE HB-O AS AN OPTION

• WHEN CHOSEN, THE HB RESULT WILL BE REPLACED WITH THE HB-O RESULT AND THE MCHC AND MCH REMOVED

• HB-O CAN BE USED TO CALCULATE THE NEW MCH AND MCHC

• AUTOMATIC COMMENT ATTACHED TO THE RESULTS STATING THAT THE OPTICAL HB IS REPORTED AND WAS USED TO CALCULATE THE MCH AND MCHC

HB-O MAY NOT MAKE US LIKE LIPAEMIA . .

BUT MAYBE IT COULD HELP US DISLIKE IT A LITTLE BIT LESS?

QUESTIONS?

REFERENCES

• SYSMEX XN 9000 OPERATOR TRAINING MANUAL. XN TRAINING MANUAL (PART

I), REVISION 1: OCTOBER 2012. ROCHE DIAGNOSTICS NZ LTD.

• WWW.ECLINPATH.COM/HAEMATOLOGY/TESTS/HEMOGLOBIN/

• BERDA-HADDAD Y, FAURE C, BOUBAYA M, ARPIN M, COINTE S, FRANKEL D, ET

AL. INCREASED MEAN CORPUSCULAR HAEMOGLOBIN CONCENTRATION:

ARTEFACT OR PATHOLOGICAL CONDITION?. INTERNATIONAL JOURNAL OF

LABORATORY HAEMATOLOGY 2017; 39: 32-41.