BLOOD FILM EXAMINATION: ITS RECENT INVESTIGATIVE METHODOLOGY IN THE DIAGNOSIS OF DISEASE

In patient’s care, diagnostic formulations rest on a tripod consisting of clinical history, physical examination and laboratory investigation.
The literature reveals that as much as 70% of clinical decisions and diagnosis are supported by laboratory medicine (WHO, 2015; Adewoyin & Nwogoh, 2014).

Peripheral blood film (PBF) is a basic and a highly informative haematological tool at the clinician’s disposal in screening, diagnosis and monitoring of disease progression and therapeutic response.
An adept understanding of peripheral blood interpretation is important for a successful clinical practice (Adewoyin & Nwogoh, 2014).

Therefore, the ability to prepare, stain and report correct findings of a peripheral blood film is a skill that every Medical Laboratory Scientist should desire and study extensively to get expertise in.
This is important as its role in the investigations and diagnosis of diseases mostly anaemia and most other haematological disorders cannot be over emphasized. To me, it is the hallmark of haematology.

1. BLOOD FILM EXAMINATION: ITS RECENT INVESTIGATIVE METHODOLOGY IN THE DIAGNOSIS OF DISEASE A SEMINAR PRESENTED TO THE DEPARTMENT OF HAEMATOLOGY FEDERAL TEACHING HOSPITAL, ABAKALIKI BY SCT. NWUDELE CHIBUEZE SCT. IWUCHUKWU CALISTUS KANAYO. DATE: 4 TH NOVEMBER, 2015 .

2. INTRODUCTION In patient’s care, diagnostic formulations rest on a tripod consisting of clinical history, physical examination and laboratory investigation. The literature reveals that as much as 70% of clinical decisions and diagnosis are supported by laboratory medicine (WHO, 2015; Adewoyin & Nwogoh , 2014). Peripheral blood film (PBF) is a basic and a highly informative haematological tool at the clinician’s disposal in screening, diagnosis and monitoring of disease progression and therapeutic response. An adept understanding of peripheral blood interpretation is important for a successful clinical practice ( Adewoyin & Nwogoh , 2014). Therefore, the ability to prepare, stain and report correct findings of a peripheral blood film is a skill that every Medical Laboratory Scientist should desire and study extensively to get expertise in. This is important as its role in the investigations and diagnosis of diseases mostly anaemia and most other haematological disorders cannot be over emphasized. To me, it is the hallmark of haematology .

3. HINTS ON BLOOD FILM QUALITY To ensure accurate and reliable results, pre-analytical variables that affect the quality of film must be controlled ( Adewoyin and Nwogoh , 2014). Blood to anticoagulant ratio should be in the right proportion. Samples are best analyzed within 2 hours of blood collection as delay in preparation of blood film may allow for the degeneration of cellular elements of blood and may result in a pseudo-thrombocytopenia (Bain, 2005). Commonly used stain in our facility is Leishman stain which is composed of polychrome methylene blue and eosin. While methylene blue stains the acidic content of the cell (nucleus), the eosin which is acidic stains the cytoplasm of the cell ( Ochei and Kolhatkar , 2007).

4. WHICH PART OF THE SLIDE SHOULD BE EXAMINED FOR WHAT? The slide is viewed at the body of the film, usually beginning about one millimeter away from the tail (the monolayer part).The head should be avoided as the cell density is twice that seen at the tail. The head portion of the film might be of interest when investigating for the presence of malaria parasite or microfilaria. The fathered end may be examined for platelet clumps and large cells like monocytes and blasts ( Adewoyin and Nwogoh , 2014).

5. smear showing a viewable side in a slide ( Slomianka , 2009)

6. WHITE BLOOD CELLS (LEUCOCYTES) GRANULOCYTES Neutrophils Eosinophils Basophils . AGRANULOCYTES Lymphocytes monocytes

7. NEUTROPHILS Size : measures 10-14 microns Nucleus : are lobulated , connected by thin strands of chromatin and stain deep reddish purple. Cytoplasm : are abundant and stains light pink. Main function : capture and destroy invading organisms and other foreign toxic materials. Reference range : Absolute Number Adults: 40-75% 1.5-7.5 x 10 9 /L Children: 20-45% 1.5-6.5 x 10 9 /L Increase is Neutrophilia as in bacteria infections Decrease is Neutropenia as viral infections

8. A stained blood film showing neutrophils ( Slomianka , 2009)

9. EOSINOPHILS Size : measures 12-17 microns Nucleus : lobulated (mostly 2 and three occasionally) and contain nucleoli. Cytoplasm : not clearly visible because it contains large round orange-red granules and occasionally vacuoles. Main function : associated with 1gE antigen-antibody reactions. Reference range : Absolute number 1-6% 0.02-0.6 x 10 9 /L Increase is eosinophilia as seen in helminth infections ( Hoffbrand , 2011).

10. A stained blood film showing eosinophil ( Slomaianka , 2009)

11. BASOPHILS Size : measures approximately 10 microns (smaller than neutrophils and eosinophils ) Nucleus : usually bilobed , and stain deep purple-blue, but obscured with cytoplasmic granules. Cytoplasm : slightly basophilic and contains large granules which stain purple or black. Main function : Phagocytes (contains heparin, histamines and serotonin). Reference range : Absolute number 0-1% 0.01-0.1x 10 9 /L Increase is basophilia as seen in myeloproliferative disorders ( Ochei and Kolhatkar , 2007).

12. A stained blood film showing basophils ( Slomianka , 2009)

13. LYMPHOCYTES Size : The small lymphocytes is approximately size of red blood cells (10 microns) while large lymphocytes measures 12-16 microns. Nucleus : Round or irregular and dark mauve staining. Large lymphocytes are sometimes indented. Cytoplasm : small lymphocytes has little (small) cyplasmic space that stains dark blue, while large lymphocytes has abundant cytoplasmic space. Main function : Both effect the immune defense system (inform of T and B-lymphocytes). Reference range : Absolute number In adults: 21-40% 1.2-4.0 x 10 9 /L In children: 45-70% 6.0-8.5 x 10 9 /L Increase is lymphocytosis as seen in protozoa infections Decrease is lymphocytopenia as seen in HIV/AIDS. ( Cheesbrough , 2010).

14. A stained blood film showing lymphocyte ( Slomianka , 2009)

15. MONOCYTES Size : measures 15-20 microns in diameter. Nucleus : Kidney shaped, stains unevenly with a stringy appearance. Cytoplasm : Abundant and stains greyish blue and vacuole may be seen. Main function : Phagocytic in function and capable of ingesting a large number of bacteria. Reference range : Absolute number 2-8% 0.2-1.0 x 10 9 /L Increase is monocytosis as seen in chronic bacterial infections. ( Adewoyin and Nwogoh , 2014)

16. A stained blood film showing monocyte ( Slomianka , 2009)

17. DIFFERENTIAL WHITE BLOOD CELL COUNT: WHAT YOU SHOULD KNOW Differential white blood cell (diff. WBC) count is performed to determine the relative number of each type of white cell present ( Cheesbrough , 2010). The white blood cell (WBC) count on its own is not very informative in evaluating the state of health of an individual. The presence of a normal WBC does not mean that all is well with a patient (Munster, 2012). In view of this, it is common practice to provide a so-called WBC differential count. The standard WBC differential divides the white blood cells into the 5 major sub-population which include lymphocytes, monocytes , neutrophils , eosinophils , and basophils (Munster, 2013).

18. OBSERVING AND RECORDING NUCLEATED RED BLOOD CELLS ( nRBCs ) If nRBCs are observed while performing the differential they need to be reported. Correct the WBC count if the nRBC count is greater than 10nRBCs/100. Use the following to calculate corrected WBC ( cWBC ) nRBC = WBC x 100/( nRBC +100) ( Constantino et al ., 2000; Adewoyin and Nwogoh , 2014 ).

19. METHODS OF DIFFERENTIAL COUNT Battement method Longitudinal method

20. PROCEDURES Place a drop of immersion oil on the lower third of the blood film and cover with a down cover glass ( Cheesbrough , 2010). Examine the blood film under the low power (x10) objective. To evaluate the quality of the blood film. To estimate roughly the red cells and white cells count ( Ochei and Kolkatkar , 2007).

21. EXAMINATION OF THE BLOOD FILM UNDER OIL IMMERSION (X100) OBJECTIVE Take the differential count leucocytes of 100-cell and record your result using manual chart format or automated cell counter. When the WBC is very low (below 1000/ uL ), it is difficult to find enough WBCs to perform a 100-cell differential. In this situation, a differential is usually performed by counting 50 cells. A notation on the report must be made that only 50 white cells were counted. Multiply each percentage by 2. When the WBC is very high (>50,000/ uL ), 200-cell differential may be performed to increase the accuracy of the differential. The results are then divided by 2 and a note made on the report that 200 leucocytes were counted. Calculate the absolute number of each white cell type by multiplying the number of each cell counted in % by the total WBC count.

22. A stained blood film showing different white blood cells ( Gillet , 2009 )

23. Observe and report the morphological abnormalities in red cells. Observe and report the abnormalities in leucocytes. Evaluate platelet count and morphology . Observe for the presence of blood parasites. Observe inclusion bodies or other abnormalities ( Ochei and Kolhatkar , 2007). BLOOD FILM REPORT

24. RED BLOOD CELL MORPHOLOGY Scan area using x100 (oil immersion). Observe 10 fields. Red cells are observed for size, colour , haemoglobin content or pallor, shape, presence or absence of inclusion bodies NORMAL MORPHOLOGY Normocytic : normal cell size and shape. Normochromic : normal haemoglobin content and colour .

25. ABNORMAL MORPHOLOGY Red cell morphology must be scanned in a good counting area. Two questions should be asked: Is the morphology seen in every field? Is the morphology pathologic or artificially induced? ( Adewoyin and Nwogoh , 2014).

26. RBCs Abnormal Morphology NOMENCLATURE OF RED CELL SHAPES NEW TERMINOLOGY OLD TERMS Discocyte Biconcave disc Echinocyte (I-III) Burr cell, crenated cell, berry cell Acanthocyte Spur cell, acanthoid cell Stomatocyte Mouth cell, cup form, mushroom cap. Spherocyte Spherocyte , microspherocytes Schizocyte Schistocyte , helmet cell, fragmented cell ELLIPTOCYTE AND OVALOCYTE Drepanocyte Sickle cell Codocyte Target cell Dacryoctye Teardrop cell, tennis racket cell    

27. QUALITATIVE GRADING OF RBC MORPHOLOGY Grade Degree of Abnormalities Marked1 to 5 cells/10 fields Slight 6 to 15 cells/10 fields Moderate > 15 cells/10 fields Grading Inclusions Rare 0 to 1/ hpf  Few 1 to 2/ hpf  Moderate 2 to 4 / hpf Many > 5/ hpf   hpf , high-power field ( Gillet , 2009)

28. A stained blood film showing red blood cells with platelets ( Slomianka , 2009)

29. WHITE BLOOD CELL MORPHOLOGY Most alteration in leucocytes morphology can be classified into three categories: Toxic or reactive changes Anomalous changes Malignant changes (Bain, 2005).

30. TOXIC OR REACTIVE CHANGES Hyper-segmentation of nucleus Cytoplasmic vacoulation Toxic granulation Double bodies Basket cells or smudge cells Turk cells Reactive lymphocytes Barr bodies Auer Bodies/Auer rods (Bain, 2005).

31. ANOMALOUS CHANGES Pelger-Huet anomaly Muy-Hegglin anomaly Alder-Reilly anomaly ( Ochei and Kolhatkar , 2007) .

32. PLATELETS Platelets ( Thrombocytes ) are approximately 2-4 by 0.5 microns in dimension (which is about a third of a normal size red cell) with coarse cytoplasmic granules ( Adewoyin and Nwogoh , 2014). They are formed from budding off of the cytoplasmic of megakaryocytes in the marrow.It is expected that we see approximately 7-15 platelets on x100 objective (Bain, 2005). A decrease in platelet count is termed thrombocytopenia. Qualitative abnormalities of platelet are termed thrombasthenia and require platelet functional studies to identify them ( Hoffbrand , 2011).

33. A stained blood film showing platelets ( Slomianka , 2009)

34. AUTOMATION IN BLOOD FILM EXAMINATION The laboratory practice of haematology has evolved tremendously over the past few decades with automated analyser generated complete blood counts (CBC) having fully replaced the original manual individual parameter assay methods (Seed, 2013). In line with the principles of good laboratory practice, standardised slide making and staining procedures will guarantee good quality peripheral blood smears. The best form of standardisation is automation (Seed, 2013).

35. SYSMEX SP-1000I™ AUTOMATED HEMATOLOGY SLIDE PREPARATION UNIT

36. FEATURES AND ADVANTAGES OF SYSMEX SP-1000I™ Improves and standardizes smear turnaround time. Rapid smear preparation with first-in, first-out slide preparation and staining. Reflexive slide preparation: applies laboratory defined criteria to prepare smears. Low sample volume requirements: onboard micro-sample mode aspirates 60 μL of sample volume to prepare and stain quality smears.

37. Uses a combination of unique slide cassette and bathless staining process. Automatically adjusts the angle, speed and blood volume based on HCT value of sample. Flexible operation and Consistently produces quality smears. Has ability to stain pre-made smears (e.g. body fluid, bone marrow samples) and can produce multiple smears automatically. In routine operations, the SP-1000i provides rapid, automated preparation of peripheral blood smears to help laboratories meet and standardize smear review turnaround times (Seed, 2013)  

38. The Sysmex HemoSlider

39. The RAL Stainer

40. Mythic 22 Full blood count Autoanalyzer.

41. HAEMATOLOGY AUTO- ANALYZER An automated system for Complete blood count (CBC). PRINCIPLE OF HAEMATOLOGY AUTO-ANALYZER The principle is based on measurement of cells in a fluidic system ( Flow cytometry ) with a complex of optical systems. Cells are counted based on their sizes, granularity and volume. Exists in different Models, Example Mythic 18 auto-analyzer, Mythic 22 auto-analyzer, Sysmex autoanalyzer and mindray autoanalyzers .

42. PRINCIPLE OF OPERATION

43. USES OF HAEMATOLOGY AUTO- ANALYZER Full Blood Count ( FBC ) Estimation of R ed C ell Indices ( MCV, MCH, MCHC )

44. CARE AND MAINTENANCE Read carefully the manufacturer’s manual and then prepare SOP for use, care and maintenance. The equipment should be operated under suitable temperature. Protect from dust by covering with its protective covering. Regular and periodic checks should be done on the working reagents. Maintenance should be done by a well trained personel

45. CONCLUSION In order to ensure that the microscopic review will provide a report that can be trusted for clinical judgement, the quality of the smear and stain must be optimal. The best way to achieve this is by means of automation of both slide-making and staining. However manual knowledge of film making, staining and report cannot be overemphasised since the efficiency of automation depend on the knowledge of the user.

46. THANKS FOR YOUR AUDIENCE

47. REFERENCES Adewoyin A. S. and Nwogoh , B. (2014). Peripheral Blood Film – A review. Annals of Ibadan Postgraduate Medicine, 12 (2): 71-79. Bain, B. J.(2005). Diagnosis from the Blood Smear. England Journal of Medicine , 353 : 498-507. Basu , S. (2005). Blood cell and bone marrow morphology. The Science of Laboratory Diagnosis, second edition. Cheesbrough , M. (2010). Blood Films. District Laboratory Practice in Tropical Countries, second edition, Cambridge University Press, pp. 319-329. Constantino , B. T. and Cogionis , B. (2000). Nucleated RBCs – significance in the peripheral blood films. Laboratory Medicine , 31 (4): 223-229. Gillet , P. (2009). Haematology White Blood Cell, Anaemia Classification. Tropical Laboratory Medicine Unit, pp. 1-52.

48. Hoffbrand , A. V. (2011). Megaloblastic anaemia . In: A. V. Hoffbrand , D Catovsky E. G. Tuddenham , A. R. Green ( eds ). Postgraduate Haematology . 6 th ed. Wiley Blackwell. Munster, M. (2013). The role of peripheral blood smear in the modern haematology . SEED haematology.Sysmex . Available at http://www.sysmex-europe.com/...SEED/ sysmex-pdf . Accessed December 12,2013 . Ochei , J. and Kolhatkar , A. (2007). Examination of peripheral blood smear. Medical laboratory science: Theory and Practical. Tata McGraw-Hill publishing company limited. New Delhi, pp. 288-302. Slomianka , L. (2009). Blue histology – blood. School of Anatomy and Human Biology. University of Western Australia. Tefferi , A. and Hanson, C. A. (2005). How to interpret and pursue an abnormal complete blood cell count in adults. Mayo Clinical Procedure , 80 (7):923-936.