Analizadores Hematológicos Flags and Troubleshooting.pdf

Analizadores Hematologicos

1. 5 Part Hematology analysers-
Flags and troubleshooting
Dr Preeti Mansukhani
Consultant, P.D. Hinduja Hospital,
Mumbai

2. Agenda
•Introduction
•Evolution of analysers
•New technologies and principles
•Newer parameters and improvements
•Limitations
•Future
•Flags with cases -RBC, Platelet count and WBC
•Trouble shooting
•Summary

3. Hematology analyzers
The workhorses of the clinical laboratory.
•5-part differentials identifying
•Nucleated red blood cell counts and immature granulocytes are
emerging as sixth and seventh parameters.

4. Evolution of the Analyzer
•The first automated cell counters came out in the 1950s based on
Coulter’s electrical impedance principle in which cells pulled through
an aperture break an electric circuit, indicating both the presence of
a cell and the size of the cell
•In the 1970s, 3-part differential leukocyte counters (for lymphocytes,
monocytes, and granulocytes) entered the market.

5. Problems with electrical impedance–
Recirculation and co-incidence
Pulse A
Pulse B
Pulse C

6. “The new technologies are great,
•SYSMEXAnalyzer may determine leukocyte differentials by
inserting a fluorescent dye into the cell nucleus and measuring how
strongly it fluoresces.( Fluorescence Flowcytometry -Sysmex –XE,XN
series)

7. ADVIA-One may alter the permeability of a cell and
see how quickly it absorbs a dye.

8. Beckman Coulter –Analysis of cells in near native state

9. Which technology is better?
•Bruce H. Davis, MD, of the Maine Medical Center Research Institute
in Scarborough, ME, said all of the analyzers on the market are
generally reliable.
•“The differences are really minor in terms of bells and whistles
that may appeal to one person or another,” he said. “The
decision typically comes down to costor someone is in a
buying group so the decision has already been made.”

10. •Nucleated RBC count
•Quantitative immature granulocyte counts
•Reticulated Hemoglobin Equivalent (RET-He),which is
used to monitor erythropoiesis and the immature platelet
fraction (IPF)
•Precision and accuracy of low platelet countshas
improved considerably for most hematology analyzers.
•Manufacturers also are improving linearity and
reportable rangeacross systems.
•High-end analyzers is counting cells from body fluids

11. Limitations
•Today’s analyzers “They’re less reliable in
what I would call the qualitative
determinations. So they can tell you it’s a
immature granulocyte, but they’re not
going to be as precise in telling you what
stage of granulocyte maturation it is.”

12. Way forward -Where the Analyzer Ends and the
Cytometer Begins
•Antigen markers such as CD4 and CD8are beginning to
appear on some analyzers
•Sysmex’sinstrument is “as close to a flow cytometer
•The line between hematology analyzers and flow
cytometers will probably continue to shift for the
foreseeable future, Davis predicted.
•“The reticulocyte count is a good example,”“First it was
manual, then it was done on a flow cytometer, then it
swung back to the automated hematology instrument
once the methodologies were made in an automated
way.”

13. FLAGS
Generated data which
is not acceptable
based on
Instrument &/or user
defined criteria
To alert

14. Computing and generation of
graphs
Each pulse is
recorded as an
oscillation, the
height of which is
proportional to
the volume and
size of the cell
Histograms
Relative frequency of cells
Size

15. Histograms
Size distribution curves are separated by flexible discriminators
The histogram curve should start and end at the base line
within the discriminators

16. RBC and Platelet Histograms
Platelets have size between 8 and 12 fL; Counted between 2 and 30 fL
RBCs have size between 80-100 fL and counted between 25 and 250
fL

17. Flagging system in 5-part

18. Not Flagged

19. Flagged

20. List of IP Messages

21. Case1

22. Case 2

23. Abnormal, RBC Abn Distribution
Case 3

24. Comparison with normal
histogram
Presence of abnormal RBC and PLT such as fragmented
RBC, anisocytosis, poikilocytosis, dimorphic population,
RBC agglutination or platelet clumps
Floating discriminator

25. Nucleated red blood cell analysis
Qualitative flagging as well as quantitative
estimation

26. Case 3

27. Automated Reticulocyte count
(RET Channels)
•Separate channel
•Enhanced precision and
accuracy
•Polymethine dye stains
nucleic acid in WBCs,
nRBCs, retics and platelets
•Size vs fluorescence

28. Reticulocyte Channel
An example of efficient
Multitasking
•Reticulocyte Count
•Reticulocyte fractions
(LRF, MRF, HRF)
•Immature reticulocyte
fraction
•Ret-He (Reticulocyte Hb
equivalent)
•Platelet-O (optical
Platelets)
•Fragmented red cells
(FRC)

29. Reticulocyte Hb Equivalent –
Ret He
Ret-He (reticulocyte haemoglobin equivalent) gives the Hb content of the freshly
produced red blood cells and thus offers real-time information on iron supply
during the course of erythropoiesis.
Ret He provides information on availability of Functional Iron
Indication
•Useful to differentiate between the two most common anaemias (iron deficiency anaemia and anaemia of chronic
disease (ACD)),
•in other words, to differentiate between actual iron deficiency and ‘functional iron deficiency’
(disturbance of iron mobilisation).
• Monitoring therapy of chronic infections or tumours
• Monitoring erythropoietin therapy and iron substitution
No other /manual method for
reticulocyte Hb !!
FDA approved

30. Suspect, Fragments?
Case 4

31. Platelets
•Impedance
•Optical (Fluorescent platelets)
•Immature platelet fraction

32. Advantages of Optical Platelet
Counting

33. Hinduja study
•Comparing platelet count obtained by
impedance, optical with flow cytometry (IRM)
•Platelet counts obtained on our automated
analysers (SYSMEX XE2100 and LH 750)
showed good correlation with the and high
degree of precision
•The impedancemethod tends to undercount
plateletsin thrombocytopenic patients
•In such cases the Optical method is superior
•Follow up is also recommended by optical
method in these cases.

35. Case 5
? Platelet count
Discrepancy between platelet count on analyser and instrument

36. Our Saviour-Platelet F
Platelet –F-10,000

37. Spurious elevation of platelet count
•extensive burns,
•extreme microcytosis(as seen in HbH disease,
microangiopathic hemolytic anemia, and red cell
fragmentation in burns),
•micro-organisms like bacteria, fungi or yeast,
hyperlipidemia,
•fragments of white blood cell (WBC) cytoplasm
in patients with acute leukemia,
•hairy cell leukemia, and lymphomas or
•the presence of cryoglobulins1
•Interferences in hemolysed sample

38. •The impedance method for platelet count estimation
at low MCV values will not provide accurate platelet
count ,instead a false high platelet countshall
potentially affect platelet transfusion decisions for patients
International Journal of Science and Research (IJSR) ISSN
(Online): 2319-7064 Index Copernicus Value (2015): 78.96 |
Impact Factor (2015): 6.391
Spurious High Platelet Count by Automated Hematology Analyzer
in Patient with Microcytic RBCS
Dr Rateesh Sareen1 , Dr Menka Kapil2
Our case –Falsely elevated platelet count ???
? Microcytosis

39. Fresh sample
Platelet impedance-11,000

40. Hemolysed sample-previous one

41. Spurious elevation of platelet count
•extensive burns,
•extreme microcytosis
•micro-organisms like bacteria, fungi or yeast,
hyperlipidemia,
•fragments of white blood cell (WBC) cytoplasm
in patients with acute leukemia,
•hairy cell leukemia, and lymphomas
•the presence of cryoglobulins1
•Interferences in hemolysed sample

42. Immature PLT are identified by its increase in
fluorescence(more RNA), FSCis also higher.
Indicator of BM function
Predictor of platelet counts in dengue patients
IPF
Immature Platelet fraction

43. WBC Flags

44. Suspect, Blast / Abn Lympho?
Case 7
Possible presence of blast,
abnormal lymphocyte or
others abnormal cell.
Abnormal clustering in
the region for blasts
and abnormal
lymphocytes

45. Newer parameters -Differential
fluorescent staining –Immature
granulocytes (Sysmex)

46. Comaprison with a normal
scattergram
Possible presence of
metamylocyte, myelocyte
and promyelocyte

47. Case 8

48. Is it enough? Labs own rules
•The manual differential is the most technically
demanding in the lab.
•Every laboratory has to determine the criteriathey
will use for what gets reviewed and what gets scanned
and generates a manual differential.
•Finn said medical directors should use the technology to
free up technologists to do more interpretive work of
cellsthat are interrogated by methods other than
microscopes, “
•The bottom line is reducing the amount of time
someone goes to the microscope as the potential is
basically accumulating behind this wave of
technology,” he said.

49. Smear review criteria for
automated CBC and diff analysis

50. Trouble shooting-
spurious results
•Inadequate blood samples, situations induced
by the anticoagulant(s)
•Technical considerationsabout performances of
the various HA
•Spurious thrombocytopenia-presence of
ethylenediamine tetra-acetic acid (EDTA) used
as the anticoagulant.
•PLT rosetting around white blood cells (WBC;
satellitism) and PLT-WBC aggregates.

52. Treatment for lipemic samples
•Lipemia interferes with hemoglobin
•Does not-RBC, WBC and platelet count.
•Correction-The first is to report only the
hematocrit, RBC, MCV, WBC, and platelet
count suggest repeating HB after the patient
has fasted at least two to three hours.
•Another approach is to perform a saline
replacement procedure.

53. •Esp. pediatric samples
•Transfer to an aliquot and run the
sample.
Beware of partial aspirations

54. •New parameters
•Increased sensitivity and precision (e.g., IGs, nRBCs).
•Parameters like Ret-Hedo not have another comparable/
manual method, making such a parameter invaluable
•IPF gives insight into the pathogenesis of
thrombocytopenia. Can help avoid unnecessary BM
examination and platelet transfusions.
•Newer parameters add a lot of extra information to the
standard CBC, which may translate into better patient
care.
•Novel Parameters still need to be standardised across
instruments and labs should make their own normal
ranges before using them
Summary