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It’s not Black and White: Unraveling the puzzles of Hematology

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Disclosures

u I am receiving an honorarium from Sysmex for preparing and delivering this presentation

u The views expressed in this presentation are those of the presenter and their healthcare facility and provided for illustration purposes only. Results of case

studies are not predictive of other cases and results may vary. Prior to using these devices, please review the manufacturer’s Instructions for Use.

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ObjectivesAt the end of this presentation, the attendee will be able to:

u List common preanalytical issues with hematology specimens

u Determine if a sample has a true abnormal value or a spurious result

u State the steps to resolving problematic samples, which include clumped platelets, cold agglutinins, critical low or high counts, and abnormal distribution/scattergrams

u Correlate cells seen on slides with numerical values of parameters

u Describe how to select, implement, and review delta check practices

u Investigate and interpret delta check alerts

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Health Care Changes

Old modelPayment for volume

More tests = lower costs = more revenue

Medically unnecessary testing was performed

New modelPayment for value

More tests, more procedures = higher costs and less profit

Motive for labs to work with physicians to develop and better utilize tests

Everything must be productive4

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Volume to Value Shift

Everything must be productive

Motive for labs to work with physicians to develop and better utilize tests

• Decreases cost• Helps physician to choose best therapy for patient

• Improves patient care• Improves outcomes• Patients are more satisfied

The right test at the right time

• Immediate access to lab test results improves patient outcomes

• Use new parameters on analyzers to help physicians in making diagnoses

• If we can detect disease earlier, easier to treat

Focus on early diagnosis

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What can labs do? Attributes of Innovative Labs

New technologies in diagnostics

AutomationUse of

middleware

New quality standard- six sigma, LEAN

Efficient workflow

Reduced turn around time

Collaborate with providers to improve patient outcomes

Eliminate ‘non value-added’ tests, methods, procedures

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How Can We Add Value??

Create

Create an efficient workflow

Encourage

Encourage physicians to order automated differential• Instrument ANC

reported with CBC w/autodiff

• nRBCs reported with the CBC w/autodiff

Use

Use the tools given to us!• Automation• Middleware• CellaVision• X BarM, BCQM • Trained

technologists

Use

Use “Best Practices”

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What are ‘Best practices’?u “A best practice is a method or technique that has been

generally accepted as superior to any alternatives because it produces results that are superior … or because it has become a standard way of doing things”1

u Best practicesu Complying with requirementsu Used to maintain quality u Can be based on self-assessment or benchmarking

u Goalsu Reduced TATu Following SOPs- the standard way of doing things!u Accurate and Precise results

1 Wikipedia

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What are Clinical Laboratory Scientists?

u CLS or MLT

u “Diagnostic detectives”

u We can’t be good detectives without looking at all the clues!

u Evaluate the results of laboratory analyses for accuracy and validity, and correlate laboratory data to disease processes

u Use Best practices!9

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Hematology is not always black and white

Standard Operating Procedures are our GPS

BUT u We can't just blindly follow

u If we just follow our GPS we don’t learn

u We need to follow SOPs, but if we don’t look at the whole picture, we could end upgoing down the wrong path!

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Hematology is not always black and whiteuStandard operating procedures (SOP)

uWhen we get a sample in hematology, we are looking at more than the results

uOperator alertsuOperator alerts give us more detailed direction

uAttention to detail

uis specimen adequate?, patient age, location, deltas,

uDeductive reasoninguWe are Diagnostic Detectives! Use All those things you learned in school!

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Instrument Flags

• No flags generated• Results may not be normal, but results usually

reported without review• Use autoverification

NEGATIVE judgement

• results may be unreliable • Confirm according to your laboratory SOP

Asterisk (*) next to a parameter

• Flags can be on Count, Morph, Diff• Recognize why the flag exists• Use histograms and scattergrams

Flags- Positive judgement

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Abnormal vs. Spurious Results

u In most cases, automated analyzers give quick and accurate results for both normal and abnormal samples

u When flags exist, investigate!

u Abnormal or spurious result?

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Abnormal resultsu True reflection of patient status

u Correlate with patient history

u May autovalidate with no flags

u Flagsu Results may be unreliable

u If flags present, Investigate

u Critical results flagged for notification

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Spurious Resultsu Erroneous results for any parameteru Not a true reflection of patient statusu Typically Pre-analytical

u Specimen collection or handling problems

u Underfilled/overfilled tube

u Clotted specimen

u Hemolyzed

u Lipemicu Wrong patient drawn ☹

u Contamination

u Analytical-Instrument problems u Reagent

u Check for instrument errors15

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Insufficient Blood Volume (short sample)

u What do you do?

a) Reset alarm, ignore message

b) Find the sample and rerun

c) Sample looks ok, must be contaminated, call floor to request new sample

d) Take a break

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OR? J

u Locate sample

u Check for clot, check volume, rerun if ok

u Visually check blood

u If suspect low hemoglobin

u Turn off aspiration sensor

u Remix & rerun in manual mode

Sysmex, 2019

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Retic Abnormal Scattergramu *Abnormal scattergram ok to report if no asterisks (*)

uYou’re done!1

u Flag indicates interfering substances

uDilute sample and rerunuDilutions >1:5 should not be used

uAdequate particles needed for accurate gating uTurn aspiration sensor off

uDiluted RBC count must not be <0.50 x 106 /µLuIf < 0.5 make lower dilution

*Sysmex users1Sysm ex XN F lagging G u ide

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Retic Abnormal Scattergram

u Dilutionsu % retic, IRF are %/ratio, RET-He measured at cellular level

u no multipliers neededu Only use multiplier for absolute

u If (*) does not go away

u Review smear for polychromasia, parasites, NRBCs, HJ Bodies, basophilic stipplinguIf present, add comment ‘may be affected by

interfering substances’u Perform reticulocyte by an alternate method

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RBC Indiciesu Maxwell Wintrobe developed first reliable

Hematocrit measurement, 19291

u Spun Hct , Wintrobe tube

u Investigated relationship of normal RBC measurementsu RBC, Hemoglobin, Hematocritu Indicies

uMCVuMCHuMCHC

u Math? ?1

1Sysmex White paper

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“Rules of 3”u Quick shortcuts

u Hemoglobin x 3 = Hematocrit +/- 3%

u RBC x 3.3 = Hemoglobin +/-1.5 g/dL

u RBC x 9 = Hematocrit +/- 3%

u Used to tell us

u Normal RBCs?

u Abnormal RBCs?

u Analytical error?21

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Rules of 3 are ‘history’!

Current technology can automatically calculate indicies

Not every sample will follow “rules of 3”

They only work when the RBCs are normal!!

Normal Hgb content

Normal size22

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Counting and sizing RBCsu Classic Impedance counting

u Curves are not Gaussianu Inaccurate pulses if cells do not pass directly though center of

apertureu Some cells pass through togetheru Some cells recirculate and are counted twice

u Impedance + Hydrodynamic focusingu Eliminates inaccurate pulses

u Directs cells singly through center of apertureu Cells remain close to normal, not deformedu Accurate determination of Hct and MCHCs in both normal and

abnormal samples

u RBC counting technology can influence MCHC results

u Curves are near Gaussian

u Curve

u Som e norm al patients can be at h igh end

11Sysm ex W h ite paper

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MCHCu 95% of normal values will be within 2SD of meanu 5% of normal healthy individuals have MCHC results <32.0 or

>36.4 g/dLu MCHC at high end of normal or slightly increased

u Normally healthy young males with Hgb at high end of normalu Low MCHC, Low MCV

u Microcytic, Hypochromic anemiau Normal MCHC, Low MCV

u Hemoglobinopathiesu High MCHC also seen in patients with HgS, HgbSC, HgbC

u Hyperdense RBCs

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High MCHC

Low Sodium affecting Hct?

Electrolyte imbalance

Lipemia, Icterus. Abnormal protein

or severe leukocytosis

Can affect Hgb

Hemolysis

Low or Normal MCV, High MCHC

Dilute 1:5 with CELLPACK DCL, correct affected results for dilution factor

Low or Normal MCV, High MCHC

1:5 dilution; Correct results for dilution factor

Lipemia or Icterus Only; plasma replacement

Low or Normal MCV, High MCHC

Spurious!Recollect sample

Did you know??

It’s not always a Cold Agglutinin!

Check Pattern of Results

Look at MCV and MCHC

Other interferences may affect Hct and cause >MCHC

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High MCHC, High MCV Cold agglutinin, RBC Agglutination, RouleauxHigh MCVHigh MCHC, MCHDecreased RBC, Hct

Scan smear for agglutination

Warm 15-30 min,mix, rerun

Dilute, or do plasma replacement if severe; incubate

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Cold Agglutininu MCHC is >37.5 g/dLu Suspect, Turbidity/HGB

Interference? u Turbidity may be present in the

diluted and lysed sampleu May Interfere with Hgb

detection and falsely increase Hgb

u Other interferences may affect Hct and cause >MCHC

u Asterisks (*) appear next to the HGB, MCH and MCHC

RT 37C

WBC 4.52 6.21

RBC 0.31 2.88

Hgb* 10.3 10.1

Hct 3.6 30.7

MCV 116.1 30.7

MCH* 332.3 35.1

MCHC* 206.1 32.9

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Note Indicies: High MCV, Low MCHCu Severe hypernatremia

u Anemias = microcytic/hypochromic cells or microcytic/normochromic

u Generally not macrocytic/hypochromic

u Indices on this patient do not make sense.

u Dilute, equilibrate for 15 minutes

u RBC measurements change

RT 37C

WBC 3.82 3.82

RBC 2.50 2.54

Hgb* 7.2 7.2

Hct 25.9 22.9

MCV* 103.2 89

MCH 28.8 31.9

MCHC* 27.8 31.41

1Sysm ex W h ite paper

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Platelet Countsu Platelets: first line of defense in controlling bleeding

u Thrombocytopenia can lead to:u Easy bruising

u Petechiae

u Bleeding

u With thrombocytopenia, platelet counts can be less reliable than with normal countsu Physicians rely on precision with very low platelet

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u Need to make informed decisions about when to treat 29

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Traditional Platelet Counting Methods

•Platelets measured by size•Large platelets can be missed•Can lead to falsely decreased counts

Optical platelet counts

•At low end, other cellular elements can be counted as platelets•RBC fragments•Schistocytes•Microcytic RBCs

•Can lead to falsely increased count

Impedance platelet counts

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Platelet Counting Methods

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• Platelet specific dye• Measures RNA of platelets• Used for plt abnormal distribution

flags or low counts• PLT-F is more reliable, counting time

6x• Eliminates errors with other methods

Fluorescent Platelet

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Platelet Scenario-platelet clump flag

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No patient history

PLT=132, clump flag, reflex PLT-

F- 132,clump flag

Slide maker alarm: short sample error

Tech 1 partial validated CBC,

left PLT for review

15 min later Tech 2 checked tube

• Short sample• Clotted

specimen

Corrected report in LIS

Time consuming phone calls

Occurrence report L

Resolve alarms before verifying

any results!

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Tricky Platelets- Follow SOPs!u SOPs can’t cover every scenario

u Our procedure is all flagged results are repeated with PLT-F, check for clot, review smear

u PLT under 100 repeated with PLT- F, checked for clot and slide review

u Even if no short sample alarm, always check tube if clump flag or according to SOP BEFORE validating

u First time patient with low normal count for no apparent reason

u IPF and MPV with *. Follow SOPs for reporting results that cannot be measured due to potential interference

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Reporting platelet counts- clumpedu Review smear under scope at feathered edge

u Giant or small plateletsu Clumpsu Fragmented, microcytic RBCsu Parasites

u “Clumped platelets, appear to be within normal range”

u “Clumped platelets, appear to be decreased”u “Clumped platelets, appear to be increased”

u Add 4th comment?u May be beneficial to estimate >100 or <100u OB, L&D, NICU, OR patients may have different critical values

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ThrombocytopeniaTrue Thrombocytopenia

u Check volume in tube

u Clot checku Review deltas

u slide review, low estimate, no clumping

u report per SOP

Pseudothrombocytopenia

u Clumping due to preanalytical error-recollect sample

u Clumping due to patient condition or therapy

u Patient may be thrombocytopenic, even after clumping is resolved

u Low PLT count without hematologic disease, family history, and/or bleeding-tendency identified

u PTCP should be considered 35

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EDTA induced thrombocytopeniau Presence of EDTA dependent IgM/IgG autoantibodies

u Causes of In vitro agglutination of platelets

u EDTA induced pseudothrombocytopenia(EDTA-PTCP ) is the most common cause. 1

u Other causes: cold agglutinins, multiple myeloma, infections, anticardiolipin antibodies, high immunoglobulin levels, abciximab therapy

u Clumping not resolved? Validate alternate anticoagulant

u Na citrate, ACD, CTAD

u Add Amikacin

u Pitfalls to manual platelet counts

1(Nakashima, 2016)

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How Else Can We Add Value to Laboratory

Tests?

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The difficulty with Differentials

u Automated vs. Manual Differential

u Which to accept and report?

u Benefits of automated differential

u The innovative lab- encourage physicians to order auto diff

u Sometimes we still need to do manual diffs!u Autovalidation allows us to spend more time on those

specimens that need it

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Manual Differentials

Manual differential traditionally performed on all samples

Labor intensive, slow, expensive

Subjective; Imprecise, Inaccurate

Absolute counts needed to be calculated

100 Cell Manual Differential

Enumerates percentage of each cell type Detects presence of abnormal cells

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How can we add value to Lab tests?u Use automated differentials and autovalidation

u Fully automated systems handling CBC, differentials, reticulocyte measurement and preparation of smearsu Less subjectiveu Less hands on timeu Improved TAT

u Auto diff can count thousands of cells!u Counting more cells gives a more accurate differential u Statistically superior

u Sysmex 6 part diff can separate IG from Neuts and bands and thus ANC can be reported at same time as CBCu Improved TATu Better patient care

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Case Study: What Would You Do??u Instrument reported ANC (IANC) = ANC 0.49 (called as

critical value)u ANC is important because risk of infection is higher when

ANC is below 500 (with chemotherapy)

u 0.5 is ‘cutoff’ used to determine if patient will get chemo

u Manual diff performed, ANC = 0.53u Discrepancy??

u How to resolve?

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IANC vs. ANCWBC = 83,000

Neut% 0.77

Lymph% 93.9Eo% 0.03

Baso% 0.02

Mono% 3.1

IG% 2.2IANC 0.64

u Tech reclassified cells in CellaVision

u 115 cells counted

Neuts 0 0%

Lymphs 106 92.2%Monos 6 5.2%

Myelos 3 2.6%

No neuts seen, so ANC is now 0!

100 more cells counted, 2 neuts seenANC = .83

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Resolving IANC Discrepancyu Important to give physicians accurate and useful information

u Watch lower counts

u With normal range IANC, close match not as critical

u ex: IANC 3.42

u Validate CBC on these and release IANC

u OK even if man diff is done and ANC = 3.0 or 4.0

u Cell confirm, if appropriate

u If manual diff matches auto diff, use auto diff

u If ‘discrepancy’, or manual diff required, count more cells!

u Manually check slide for neutrophils44

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Use Technology: CellaVisionu Manual Diffs laborious and time consumingu CV Automation saves time finding cells,

simplifiesu Counts 115 cells u If very low WBC, putting same slide on

counts same cells againu Can make additional slides and merge

u If unusual cells seen, check previous diffs in CVu In LIS you don’t see cells

u Pictures in CV!

u Can see path reviews, actual cells reviewedu Use cell confirm if appropriateu Know when to reclassify and perform manual

differential

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Tools & Resources: CellaVisionu Cells can be enlarged for better viewingu More experienced techs can review a slideu Share common database across sites

u Remote accessu CV allows collaborationu Share CV across sitesu Can have pathologist review slides without transporting slides

Other benefitsu Barcoded samples reduce errors

u Better ergonomicsu Can use as a teaching tool!

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Smudge cells: Artifact or clinically significant?

u “basket cells”, Gumprecht shadows

u Remnants of leukocytesu No cytoplasm,

sometimes all that can be seen are smashed nuclei

u Formed from leukocytesu fragile

u typically lymphocytes

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Smudge cells

u Originally thought to be artifact of slide making

u Number of smudge cells seen may be clinically significant

u Not diagnostic of CLL, but …

u Newly diagnosed CLL, larger % of smudge cells is a better prognostic factor

u Vimentin is protein important in lymphocyte rigidity

u Low vimentin can mean more smudge cells and better survival rates

u Presence of smudge cells should not be ignored

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A Differential has 3 parts!

WBC differential

RBC morphologyCV Pre-classifies RBCs based on size, color, shape into 6 morphologies

Platelet estimate

Must address all 3 tabs before you can sign out slide

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New Resource-Automated Advanced RBC Morphology Application

Adds consistency to RBC morphology

Less subjectivity

Time saving

Semiquantitative and quantitative

Auto locates and presents images of RBCs

Pre-classifies RBCs based on size, color, shape, 21 morphologies

Displayed as overview and individually

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Improving Lab Operationsu CBC and diff are the highest volume tests in clinical lab

u Creating good workflow is vital for efficient lab operationsu SOPs ensure everyone does things the same way

u Less subjectivityu Use resources

u Middleware or onboard rules-Use rules and Op alerts to make things consistent

u Use Automation and Technologyu Teamwork- flags, alarms, communicationu Review instrument features and continual training

u Trained techs are one of our best resourcesu Use interesting or problem samples as teaching

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“

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It is by riding a bicycle that you learn the contours of a country best, since you have to sweat up the hills and coast down them. Thus you remember them as they actually are, while in a motor car only a high hill impresses you, and you have no such accurate remembrance of country you have driven through as you gain by riding a bicycle.

Ernest Hemingway

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Thank you!Questions?

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