RBC

How is the Erythrocyte Sedimentation Rate measured in the iSED analyzer?

The machine uses quantitative capillary photometry (aggregation) to measure ESR

→ This is faster than traditional method as it captures kinetics of RBC during the lag or the Rouleaux formation phase.

What is ESR used for? Why is it helpful?

• Helps to reveal inflammatory activity and in monitoring the progress of conditions associated with acute and chronic inflammation, including infections, cancers, and autoimmune diseases

• Especially useful for patients with unexplained symptoms and when specific diagnosis is not available effectively using other tests.

• Provides valuable information especially in conditions like temporal arteritis, polymyalgia rheumatica, giant cells arteritis, inflammatory arthroplasties, etc.

WBC Abn Scattergram:

1) How does an abnormal WBC scattergram present?

2) What are the possible reasons for this?

1) Clustering in the WNR or WDF scattergrams

2) This is nonspecific and may be due to:

• Increased numbers of abnormal cells

• poor separation of WBC/nRBC populations or different subpopulations

• Unlysed RBCs

• High numbers of PLT clumps

• Possible interfering substances or conditions

WBC Abn Scattergram Workup:

1) If the reflex and initial DO NOT match, what should be done?

2) What should be done if results have an asterisk?

3) What if results show dashes instead of numeric data?

4) What should be done when there are >5% micromegakaryocytes or uncounted RBCs?

1) Check for specimen clots and perform Manual diff and estimates as needed

2) Scan the slide and estimate for WBC, nRBC, and PLT; perform MD as needed

3) Perform WBC, nRBC, and PLT estimates as well as MD; Report out the parameters with dashes

4) The WBC count should be corrected

→ (TNC-N x 100) / (100 + #nRBCs)

What does the WBC Suspect flag indicate?

There is unacceptable gating between the populations of basophils and nRBCs.

Asterisks will be present on these cells

WBC Suspect Work-Up:

1) what is done first when the reflex test results still show flags?

2) if when reviewing the slide, there are more than 5% micromegakaryocytes or uncounted nRBCs - what should be done?

1) scan or perform MD to check for WBC and nRBC

2) the WBC count should be corrected

What is indicated by the Difference between WNR and WDF message?

This is generated based on the ratio of TNC-D to the TNC-N-.

When TNC-D/TNC-N ratio is more than 1.3 or less than 0.77

Difference Between WNR and WDF Message Work-Up:

1) what should be done first?

2) what should be done in the presence of more than 5% micromegakaryocytes?

1) perform scan or MD to detect abnormalities

2) perform corrected WBC count

nRBC Present:

1) What does this message mean?

2) Is the WBC count generally affected?

1) When there are > 3% nRBC

• It does NOT indicate that the WBC count is unreliable

2) usually not required unless flagged; perform MD if indicated and corrected WBC count as usual

RBC Abn Distribution:

1) When is this message generated?

2) What’s considered abnormal MCHC? What does this indicate?

3) If the RBC histogram presents with dual population (multiple peaks), what should be done?

1) When the histogram pattern from the RBC channel is abnormal or when RBC < 0.50 × 10^6 /microliter

2) When MCHC is less than 28.0 or greater than 37.5 g/dL

• Due to interfering substance or condition

3) Report

RBC Agglutination:

1) How is this error message determined?

2) What is the First Step?

3) What is the Second Step?

4) What if these steps dont work and MCHC does not correct due to strong cold agglutinin?

5) What about agglutinin due to warm-reacting antibody?

6) How should the slides be prepared?

7) If warming did not remove the RBC agglutiation message, what should be done?

1) Asterisks may appear next to the RBC indices

2) Check the sample for clots; recollect if clotted

3) Warm an aliquot of the sample at 37 C for 15-30 minutes then re-analyze in manual mode

4) Plasma replacement using warm CELLPACK DCL to reduce the interference from the Ab

5) Plasma replacement with room temperature CELLPACK DCL

6) Make one RT slide (to report extent of agglutination) and one 37C slide (to report diff, morphology, WBC, PLT estimates)

7) Perform 1:3 dilution with CELLPACK DCL

• allow to equilibrate for 10-15 min. at RT

• Thoroughly mix and re-analyze in manual mode with aspiration sensor OFF

• If indices have corrected, multiply WBC, RBC, HGB, PLT, and RET# by dilution factor (3)

When MCV >105 fL and MCHC <28.0 g/dL:

1) What are the possible causes of pseudomacrocytosis?

2) What is the Workup for these cases?

1) Clots, agglutination, rouleaux, normal in newborns

2) Check for clots, workup for agglutination, workup for rouleaux

Turbidity/Hgb Interference (MCHC > 37.5 g/dL):

1) How is MCHC calculated?

2) How are the indices affected?

3) What are the possible causes of turbidity?

4) What conditions interfere with the hematocrit?

5) What major RBC disorders are associated with hyper-dense RBCs with increased MCHC values?

1) MCHC = Hb x 100 / HCT (%)

2) Hb is falsely increased or HCT if falsely decreased due to turbidity interfering with light path detection

3) Due to lipemia, icterus, hemolysis, and severe leukocytosis

4) Due to plasma electrolyte imbalance, hemolysis, RBC agglutination and rouleaux

5) Hereditary Spherocytosis and certain hemoglobinopathies

→ due to altered surface volume and/or deformability of the RBCs

→ these are irreversible

Turbidity/HGB Interference MCHC > 37.5 g/dL:

1) How does low sodium and precipitation of abnormal plasma protein affect the MCHC?

• What is the Workup for this case?

1) Falsely increase the MCHC

→ water flows out of the RBC due to osmolality difference

→ abnormal protein causes spectral interference

• Make 1:5 dilution with CELLPACK DCL

• Allow dilution equilibrate for 10-15 min. at RT

• Re-analyze with aspiration sensor turned off

• If indices have corrected, multiple the WBC, RBC, HGB, HCT, PLT, and RET # by the dilution factor (5)

• If the indices have not corrected, perform a spun hematocrit use this value to recalculate the MCV and MCHC

Lipemia, Icterus:

1) What is the workup for this case?

2) How should the results be reported?

3) what should be done if TPN contamination is suspected?

1)

• Perform plasma replacement with CELLPACK DCL

• Centrifuge an aliquot of blood in a Wintrobe tube at 3000 RPM for 10 min to separate the plasma from the cells (mark the level of the blood)

• Using a pasteur pipette, remove as much plasma as possible without disturbing the buffy coat

• Add CELLPACK DCL to the marked line

• Re-suspend the red cells thoroughly in CELLPACK DCL and transfer into a glass tube

• Re-analyze mixed sample in manual mode

2) From a lipemic specimen, retain WBC, RBC, HCT, MCV, and PLT BUT use the HGB from the plasma replacement specimen to reacalculate the MCH and MCHC

→ only report HGB, MCH, and MCHC if acceptable results; otherwise report as lipemic specimen unable to report

3) Re-collect specimen

Severe Leukocytosis:

1) What is the Workup for this case?
2) What if the indices have not corrected?

1)

• Make a 1:5 dilution with CELLPACK DCL

• Equilibrate at RT for 10-15 min

• Re-analyze

• If indices have corrected, multiply WBC, RBC, HGB, HCT, PLT, and RET # by the dilution factor

2) The RBC count needs to be corrected and indices are re-calculated:

• subtract the WBC count expressed in millions from the RBC count

• perform spun HCT and use that to calculate cMCV and cMCH and cMCHC

Hemolysis Work-Up:

1) what should be done first?

2) how does the specimen look?

3) what are the next steps?

1) Perform Manual HCT and observe plasma for coloration for hemolysis

2) bright red plasma indicates in- vitro hemolysis while brownish red plasma indicates intravascular hemolysis

3) record HCT and alert supervisor/pathologist

→ can report WBC and HCT

→ report PLT-F count if possible or perform manual estimate

→ do not report other parameters and report/indicate hemolysis in specimen

MCHC < 28.0 Work-Up:

1) which RBC indices are considered to be most affected?

2) what are the possible causes for this?

3) How does severe leukocytosis lead to decreased HCT?

4) How does osmotic matrix effect cause low MCHC?

5) what should be done with samples that that exceed 350 mOsm/kg?

6) after dilution, what parameters should be corrected if reporting?

1) falsely increased HCT

2) severe leukocytosis and osmotic matrix effect

3) severe leukocytosis can actually affect both HCT and Hb due to the turbidity causing interference in light detection path

• can also cause increase in RBC count which can then cause increased HCT

4) osmotic matrix effect may falsely increase MCV due to electrolyte imbalances, hyperglycemia, drugs

• This will cause MCHC to falsely decrease due to relative hypotonic solution compared with the hypertonic cell concentration

5) if pathologically high:

• dilute sample (1:5)

• Incubation for equilibriate

• Warm at 37 C

6) multiply the DF with WBC, RBC, Hb, HCT, PLT, RET before reporting

Suspect Fragments:

1) What is indicated by the Suspect Fragments flag?

2) when PLT accuracy cannot be guaranteed via impedance due to interfering substances, what does the analyzer do?

3) How does the PLT-F channel work?

4) How should these results be reported?

1). This is determined from RET scatter gram and/or by calculation and size comparison of certain RBC and PLT items

• sometimes may cause RBC, MCV, MCH, MCHC, and RDW to have asterisk

2) Reflex to a new Fluorescent channel and increase counting time by six-fold

3) Platelets will be identified and counted using platelet-specific fluorescent dye (oxazine)

• this stains the Rough ER and mitochondria

• Correlation with CD41/61 stained plts

• Minimizes interference by RBC/WBC fragments and microcytic cells

4) PLT count may be reported if flags have been corrected, otherwise:

• Scan or enter p to match manual and automated count

• Omit any indicated values if any

• Indicate any abnormalities in RBC, WBC, PLT morphology

Abnormal RET Scattergram:

1) what is indicated by this flag?

2) what are the possible causes?

3) how should the RET parameters be reported?

4) what is the work-up for this?

1) increased activity in the RET-THR of RET scattergram or increased RET-UPP of RET-EXT scattergram

2) due to the presence of nRBCs, H-J bodies, or parasites

3) if asterisks appear, the results need to be investigated. Otherwise report if no flags

4) work-up includes:

• 1:5 dilution

• Check if RBC count matches with undiluted sample

  • Diluted RBC count should be greater than 0.50E6, otherwise increase RBC count by making lower dilution (1:2)

• If flag is corrected, multiply abs retic count by DF

• If flag not corrected, perform manual retic count using MB stain

Difference Between RBC and RET. Check the Results:

1) what does this flag indicate?

2) what are the possible causes?

3) what is the work-up for this?

1) the ratio of the RBC-O to RBC is greater than 1.2 or less than 0.8

2) this can be due to abnormal RBC morphology such as:

• rouleaux

• Agglutination

• Polychromatic

• Parasites

• nRBCs

• H-J bodies

3) start by re running the sample, otherwise:

• warm at 37

• If still not corrected after this, perform and report only manual retic count via MB stain

Suspect sample, check the sample:

1) what does this flag indicate?

2) what is the work-up?

3) how should the results be reported?

1) sample issues; based on algorithm from WNR scattergram

2) check for:

• QNS

• Clots

• If tube is overfilled

3) if flag is corrected and results are consistent, then results may be reported

• otherwise, review smear for possible interference

• Recollect if necessary

Insufficient Blood Volume:

1) what does this flag indicate?

2) what is the work up?

1) QNS detected by the sample aspiration sensor

2) check for volume and clots then remix and rerun

• if sample is suspected of having low Hb, repeat sample in manual mode

Abnormal PLT Distribution:

1) what is the work-up?

2) what should be done if platelet clumps have interfered?

3) what if vortexing does not remove the flags and enter p is not possible?

1) check for volume and clot then:

• recollect if clotted

• Check if PLT-F is consistent and flags are corrected, then file results

• If not corrected still, perform smear review for clumps, giant platelets, satellite, and fibrin strands

2) vortex an aliquot of the sample then run in manual mode in PLT-F

• report if results are corrected

• can also perform enter p

• Include comment to indicate inaccuracy due to PLT clumps

3) unable to report due to PLT clumps

• remember to omit any dashed PLT parameters

Abnormal PLT Scattergram:

1) what does this flag indicate?

1) when clustering in the PLT and IPF area on the PLT-F scattergram is abnormal

Difference Between PLT and PLT-F. Check the results:

1) what does this flag indicate?

2) what is the work up?

3) how should the results be reported?

1) ratio of PLT-F to PLT channels is more than 2.0

2) check specimen volume and clot first, then:

• rerun sample

• Scan smear for abnormal morphology of PLT

3) report if results are consistent and flags corrected, otherwise:

• enter p and scan smear

• Vortex if necessary

• Comment/indicate if necessary

• Recollect if necessary

Cold Agglutinins:

1) What indices are falsely elevated due to this?

2) What antibodies are usually associated?

1) falsely elevate MCV and MCHC on automated intstruments

2) IgM cold antibodies:

• anti-I of mycoplasma pneumoniae

• anti-i of Infectious mononucleosis

Rouleaux:

1) what causes this?

2) how do dilutions in instruments affect this?

1) caused by paraproteins decreasing the zeta potential around erythrocytes, which results in them stacking together in a column

2) in instruments with relatively small dilutions, the diluting reagent may not dilute the para proteins to permit the RBCs to separate from each other

• while larger dilutions help to prevent rouleaux

Microclots:

1) In general, what causes these to form?

2) what results are affected?

1) poor specimen collection techniques

2) falsely elevated MCV due to fibrin-trapped RBCs that adhere to one another

Osmotic Matrix Effect:

1) What are the possible causes?

2) What results are affected?

3) How are the RBCs affected?

1) causes include:

• markedly elevated blood glucose

• Electrolyte imbalance

• Patient treated with hydroxyurea

2) falsely increased MCV

3) the cells are either swollen or shrunken in the abnormal native plasma

→ these cells may not have sufficient time to presume their normal size upon exposure to cellpack dcl before sizing measurement is performed

Fragile Leukocytes:

1) How is the WBC count affected?

2) What causes this?

3) what is the work-up for this?

1) spuriously low WBC count due to increased fragility

2) this can be due to disease and/or force exerted as WBCs pass thru the aperture

• commonly found in CLL or drugs which also results in uremia

3) perform peripheral smear count to correlate with instrument count

• if this does not match, use hemocytometer count

• Use PLT-F to eliminate interference from WBC cytoplasmic fragments

• Verify via smear review

Giant Platelets:

1) What are these?

2) How should these be dealt with?

1) these are plts with size of RBC or larger; these may be granular or agranular

• falsely increase RBC count

• Falsely decrease PLT count

2) use PLT-F to reduce interference from these giant plts

Platelet Satellism:

1) what happens here?

2) what results are affected?

1) abnormal platelet adherence to the periphery of neutrophils

• due to IgG or IgM agglutinins

• Common in autoimmune disorders, pregnancy, Behcet’s disease

2) falsely decreased PLTs