1. Validation procedures for cell analyzers
Dr Archana Vazifdar
Dept. of Hemato-Pathology,
Super Religare Laboratories Limited, Mumbai 1

2. Principles of automation
Impedance – count and size cells by change in resistance produced as they are suspended in an electrically conductive medium
Optical scatter- measures scatter properties of cells by laser light
Single angle/ Multi-angle scatter

3. RBC & Platelets measured in one channel
RBC volume > fl
Platelet volume 2-20 fl
Hb & WBC measured in second channel
DLC in third channel
RBC-Plt- separated by Volume & refractive index based counting/sorting
MCV does not exceed 150–160 fl and, as there is no
particle above that size in the blood stream in health
or in disease.

4. Interpretation of data
3 steps for performing a CBC- interpretation of numeric, graphic & flag data, Delta checks, Review of peripheral smear

5. Normocytic Normochromic
RBC count
Spurious increase:
Giant PLT
High WBC counts (>50)
Spurious decrease:
Cold /warm agglutinins
Very small RBC
Cryoglobulins
As cell passes through an orifice, the change in resistance produced is measured as a single puls
No. of pulses= no. of cells
Size of pulse= volume of cell
All info is then depicted on a histogram
RBC histo- X axis, volume, Y axis, No. of RBC, MCV- mean of distribution curve

6. Platelet count Spurious increase: RBC/ WBC fragments Cryoglobulins
ADVIA 120
COULTER
Platelet count
Spurious increase:
RBC/ WBC fragments
Cryoglobulins
Lipids
Spurious decrease:
Platelet clumps
Giant platelets
CELL-DYN

7. WBC (FCM) Impedance- VCS Baso,mono, eos, blasts neutro lympho
The traditional microscopic method based on the count of 100 cells has 3 types of error: statistical error, distributional error owing to unequal distribution of cells in the smear, and error in identifying cells related to the subjective interpretation of the examiner. Newer analyzers analyze thousands of cells per sample and can produce morphologic and quantitative flags, which have significantly reduced error and allow reliable absolute counts at low and high concentrations.
WBC analyzed by fcm, using impedance (VCS), Optical scatter (cytochem)
VCS- Simultaneous measurement of volume, conductivity and scattered laser light, CELLS R ANALYZED IN NEAR- NATIVE STATE. x-axis represents laser light scatter, the y-axis volume and the z-axis conductivity
Normal WBC histogram
Normal WBC scatterplot

8. Optical scatter: ADVIA120 DLC by Peroxidase method
Spurious increase
PLT clumps & large platelets
Nucleated red cells
Resistant RBC’s
Spurious decrease:
Clotted sample
Fragile cells- CLL
Lymphoid aggregates- UTI, B- cell NHL, CMML
Storage associated degeneration
EDC include immature or atypical cells such as blasts, IGs, atypical lymphocytes, hematopoietic progenitor cells (HPCs), and NRBCs.70 The principal aims of the EDC are to further reduce the need for microscopic revision, to obtain more precise and accurate counts
when elevated basophil/ monocyte counts are produced, they must be examined with caution because they can be artifacts due to the presence of abnormal cells such as blasts, plasma cells, and lymphoma cells.

9. Flags
A signal to the operator that the analyzed sample may have a significant abnormality/ does not meet acceptance criteria/ cannot be displayed
Cause of errors:
Analyzer
Sample
Random run error

10. RBC flags Suspect flags N’rbc, R’rbc, Micro RBC, RBC fragments,
interfere with WBC & platelet counts
H & h errors
short sample, aged sample
Definitive flags
Anemia, anisocytosis, microcytosis, macrocytosis, poikilocytosis
Erythrocytosis

11. Anemia, Microcytosis, anisocytosis
Hb 8.5
RBC 3.2
FLAG:
Anemia, Microcytosis, anisocytosis
Left shift of curve:
Microcytosis
Iron Deficiency Anemia
β thalassemia trait
Anemia of chronic diseases
IDA & ACD- NORMAL RDW

12. s/o Iron Deficiency Anemia Advise Iron studies
ACTION:
RBC indices
Mentzer’s index (MCV/RBC)=
MI ≤ 13- BTT, ≥ 13- IDA
Conclusion:
s/o Iron Deficiency Anemia
Advise Iron studies

13. N’rbc, Micro RBC/ RBC fragments Giant plt Thrombocytopenia
Hb 6.4
PLT 140
MPV 7.9
PCT .148
PDW 15
Flags:
N’rbc, Micro RBC/ RBC fragments
Giant plt
Thrombocytopenia
FRBCs are identified only on the basis of size and hemoglobin content, independent of their shape
Lt of curve not touching baseline:
Noise
Schistocytes &/ extremely small rbc
Giant platelets

14. Hemolytic anemia Action: RBC Indices- MCV, RDW
PLT Histogram- MPV & PDW
Review PS- RBC morphology
-PLT count (100)
Conclusion:
RBC count falsely ↓
Platelets falsely ↑ (mask t’penia)
Hemolytic anemia

15. Dimorphic RBC population, anisocytosis
Hb- 8.6, MCH- 26.5, MCHC- 32.2
Flags:
Dimorphic RBC population, anisocytosis
Bimodal peak:
Dimorphic RBC population
Transfused cells
Combined deficiency
Therapeutic response in IDA
NN anemias: Hypoplastic, malignancy, Sideroblastic (low retic)
Acute bld loss, Sev hemolysis, AIHA, G6PD, Sickle,
Action:
Review PS to identify cause

16. 50/ F, Hb-8.9, MCV-73, MCH- 25.6, RDW-26.8 Blood transfusion
Volume/Hemoglobin Concentration (V/HC) cytogram hemoglobin concentration (chromia) is plotted along the x axis and cell volume/size is plotted along the y axis, Nine zone grid, central zone NN
Blood transfusion

17. Hb-5.1, MCV-96.7, MCH- 29.6, MCHC-31.4, RDW-24.5 TLC/Plt-Normal
45/F, Severe pallor
Hb-5.1, MCV-96.7, MCH- 29.6, MCHC-31.4, RDW-24.5 TLC/Plt-Normal
S. Fe- 25
TIBC- 144
S. Fe saturtn- 20.8
S. B
Dual/Combined deficiency

18. H&H error, N’rbc, dimorphic reds Anemia, macrocytosis, anisocytosis
Spurious ↑MCHC:
Flags:
H&H error, N’rbc, dimorphic reds
Anemia, macrocytosis, anisocytosis
Sample related problems- turbidity-↑ Hb
Lipemia/ TPN
Cryoglobulins
Autoagglutination
Hemolysis (in-vitro/vivo)
Spurious ↓ Hct
Clotted sample
Right portion of curve extended:
RBC agglutination
N’rbcs
Leukocytosis

19. Review PS: L/F agglutination vs n’rbc’s
Action:
Review PS: L/F agglutination vs n’rbc’s
After warming in H2O 37ºC for 15 mins
corrected
spuriously low RBC counts and to abnormally high MCV (each small RBC clump is considered as one single particle Haematocrit (RBC · MCV) is erroneous and spuriously low, contrasting with Hb that is measured after RBC lysis and is unaffected by agglutinins.
As a rule, the MCHC is spurious, usually >36 g/dl.
Conclusion:
False ↓ RBC, Hct,
False ↑ MCV, MCH & MCHC
Cold agglutinin disease

20. Causes of H&H mismatch:
partial sample aspiration/ improper mixing
Hb/ MCV measurement error/ very low
High WBC counts (interfere with Hb measurment)
Cold agglutinins
Short sample (microtainer)
Repeat collection

21. Platelets Smallest guys largest culprits!!
As platelet counts fall, reliability of analyzer decreases.
Conventional methods are unable to provide consistently accurate results in lower range
Clinicians using thresholds of 5-10 X 109/l must be aware of the limitations in precision and accuracy of cell counters
Linearity : 10–1,000 X 109/l
Thresholds for use of prophylactic plt tx has reduced frm 20 ×
103/µL to 10 × 103/µL (20 × 109/L to 10 × 109/L). Other
Authors have suggested that in patients without fever
or bleeding, there may be even lower values. However, the
utilization with confidence of these new thresholds requires
knowledge of the limitations in precision and accuracy of the
analyzers at these count levels.

22. Common platelets flags
PLT Clumps
↓Plt counts
Interferences with WBC Results (↑WBC counts)
Giant platelets
Small platelets
PIC/POC delta- difference > 20,000
Thrombocytopenia- true/false

23. Flags:
Small platelets
Increased small sized particles:
Noise, debris, lipids, bacteria, fungi
? Wiskott Aldrich syndrome
Debris/ noise
Conclusion:
Falsely elevated platelet counts

24. Falsely ↓ Plt count, ↑MPV
Flags:
Giant platelets, platelet clumps
Cellular interference
Action:
Review PS for platelet count
Non fitted curve with increase in large cells:
Large platelets, clumps
Conclusion:
Falsely ↑RBC count
Falsely ↑WBC count
Falsely ↓ Plt count, ↑MPV
Giant platelets

25. 45/M PIC/POC delta Excessive noise included in impedance count
Debris, bacteria, fungi
Plt clumps
Giant plt
CAUSE- Plt clumps, giant plt, ….?

26. WBC Flags IG, Band, Blasts Aty ly, Variant ly MPO, non viable WBC
N’RBC, rst RBC
Plt clump
Outside Reportable Range
Leukocytosis, monocytosis, basophilia, eosinophilia
Unable to Find Clear Separation between WBC subpopulations

27. Shoulder on the left of curve: N’rbc Lyse resistant RBC
Platelet clumps/ Giant platelets
Fibrin
Impedance noise
N’RBC: neonatal hemolytic disease, in premature neonates and neonates affected by hypoxia in the perinatal period.
adults: thalassemic syndromes, myeloproliferative diseases (myelofibrosis), bone marrow metastases of solid tumors, extramedullary hematopoiesis, and all of the conditions of hematopoietic stress (eg, septicemia, massive hemorrhage, and severe hypoxia)
Resistant RBC’s: physiological (neonates) pathological (abnormal hb, liver disease, uraemia, chemotherapy)
extended lysis mode

28. CML Flags: IG, Blasts, eosinophilia,monocytosis, lymphopenia
Leukocytosis
Thrombocytosis
Anemia

29. Acute Leukemia Flags: Aty lymphocyte, Variant lymphocyte
Non-viable wbc
Leukocytosis
T’penia
Acute Leukemia

30. 38/F, k/c/o DM Plt 100 Flag: leukocytosis, n’rbc, dimorphic reds
it is useful not only to identify the presence of NRBCs, but also to estimate the NRBC count: 1.to evaluate the efficacy of transfusion therapy, as with thalassemic syndromes in which it is advisable to maintain an NRBC concentration of less than 5/100 WBCs
2. persistence of NRBCs in the peripheral blood of subjects undergoing stem cell transplantation has been shown to be a poor prognostic factor
3. hospitalized patients after general or cardiothoracic surgery or with other nonhematologic disease, the mortality rate was 21.1% for patients with NRBCs, whereas it was 1.2% for patients without
Flag: leukocytosis, n’rbc, dimorphic reds
Conclusion:
21 nrbc’s/100 wbc- corr WBC= 17.35
DM in sepsis with liver abscess

31. VCS-Neutrophil population data
Newer Aspects:
VCS-Neutrophil population data
VCS:
Quantitative
Operator independent
Routinely available
Inexpensive
INCREASE MEAN NEUTROPHIL VOLUME (MNV)
DECREASE MEAN NEUTROPHIL SCATTER (MNS) – left shift
Lacking leukocytosis or neutrophilia
Suggestive of acute bacterial sepsis

32. Automated malaria detection
“Gold standard” - thick & thin smear
Need for rapid, sensitive & cost-effective screening technique
Hemazoin pigment
Activation of neutrophils & monocytes
Increase volume heterogeneity (anisocytosis) of monocytes & lymphocytes, detected by VCS
‘Positional parameters’, used as objective criteria for detecting presence of plasmodium
Clin. Lab. Haem., 26, 367–372 Automated detection of malaria

33. Vol SD lymphocyte X SD Monocyte / 100 > 3.7
Plasmodium falciparum
Normal
Monocytes
Reactive LY
Parasitized RBC
shoulder
Vol SD lymphocyte X SD Monocyte / > 3.7
Am J Clin Pathol 2006;126:
Briggs et al / MALARIA DETECTION USING VCS TECHNOLOGY

34. Specificity is 94% and sensitivity 98%
PPV is 70% and NPV 99.7%.
A flag indicating potential presence of malaria is a valuable diagnostic method for detection of malaria and may become a routine parameter in it’s diagnosis

35. Reticulocyte Indices
most promising from a clinical viewpoint are the CHr and the MCVr.
CHr:
directly reflects hemoglobin synthesis in marrow, & measures iron availability.
↓ IDA & BTT (independent of iron stores)
MCVr: ↑rapidly following iron therapy
↓ with the development of iron-deficiency
↓ in macrocytosis after therapy with B12 &/or folic acid
Available in very few analyzers, not standardized
CHr- reduction indicates iron-deficient erythropoiesis, even in conditions in which traditional biochemical markers such as ferritin and transferrin are inadequate (eg, in cases of inflammation or
anemia from chronic disease), and, on the other hand, it is useful for monitoring early response to intravenous iron therapy because it increases significantly after only 48 hours. exceptions are heterozygotes for β-thalassemia whose CHr is always reduced independent of iron stores.

36. Case 1
38/M, No history available

37. Cold agglutinin disease
Result after treatment in H ̊C
Cold agglutinin disease

38. 27/M, Hb 7, MCV 94, MCH 32, MCHC 35.7, RDW 14.6, Plt 158
Case 2
Flags: Blasts, IG, n’rbc, rbc fragments, giant platelets

39. 50 nrbc’s/100 WBC
Spherocytes +
Giant platelets
Conclusion:
Severe hemolysis following Primaquine ingestion in G6PD deficiency

40. Case 3 : 33/M, Thrombocytopenia X 6 mnths, no bleeding
Case 3 : 33/M, Thrombocytopenia X 6 mnths, no bleeding. All other parameters WNL, ? ITP
Flags: n’rbc, micro rbc/ rbc fragments

41. Action:
Change anticoagulant to Sodium Citrate
Platelet count- 243
Conclusion
EDTA dependant pseudothrombocytopenia
(EDP)

42. EDP EDTA dependant pseudothrombocytopenia (EDP):
Hypothesis- antigen-binding site in the GPIIb/IIIa complex , normally hidden/cryptic, is modified by or exposed only in presence of EDTA
In-vitro phenomena
Associated with autoimmune/ neoplastic pathology, but also seen in healthy individuals
Abnormal plt from CMPD, more prone to clumping by EDTA
Alternate anticoagulants; 10% trisodium citrate/ ACD

43. Case 4: 15/M, Fever

44. Malaria discriminant factor= 6.3
Conclusion:
Plasmodium falciparum , PI 15%
Thrombocytopenia

45. THANK YOU
Archana Vazifdar, M.D.
SRL RELIGARE LTD.