Plasma fibrinogen in Recognizing Buffalo Inflammatory Disease: A Retrospective Study of 21 Cases

Dinesh Kumar SinghAssistant Professor, Dept. of Veterinary Pathology and Clinics, IAAS, Nepal

AbstractA retrospective study was conducted to determine how useful fibrinogen is in

detecting inflammation in buffalo. The study analyzed 21 sequential leukogram with available fibrinogen data. All samples were from various clinical entities, which were collected from the field during the period 2003 to 2005. Leukograms were categorized as normal, stress, or inflammatory. Corresponding fibrinogen values were categorized as normal (200 to 800 mg/dl) or increased (greater than 800 mg/dl). The fibrinogen values were compared with the classification of the leukogram. Plasma protein/fibrinogen rations were calculated for all samples. Values less than 10 were classified as inflammatory, ration of 10 to 15 were considered to be suggestive of inflammation and ratio greater than 15 were considered normal. Of all leukogram evaluated, 23.81% (5 of 21) were categorize as normal, 9.52% (2) as stress, and 66.67% ((14) as inflammatory. Of all leukogram, 52.38% (11) had elevated fibrinogen levels. Of all hemograms associated with normal leukogram, 40% had elevated fibrinogen levels. Of all stress leukogram, 50% had elevated fibrinogen. Of all inflammatory leukograms, 64.3% had elevated fibrinogen. 42.8% (3 of 7) of all buffalo with stress or normal leukograms had pp/fib rations less than 10, which would indicate inflammation. 35.71% (5 of 14) of buffaloes with inflammatory leukograms had no clear evidence of inflammation based on pp/fib rations of 10 to 15 or greater than 15.

IntroductionIt is very important to recognize inflammatory disease in a patient for treatment.

The ability to characterize the inflammation in term of origin, severity, and progression allows veterinarian to choose the best therapy and to monitor its results. To recognize the inflammatory disease, we usually depend on the interpretation of clinical signs with combination of hematology and clinical biochemistry abnormalities.

If there is big abscess or purulent exudative wounds, there are no big diagnostic dilemmas and veterinarian usually is confident with the clinical assessment of the situation. We don't need complete blood cell count and fibrinogen determination to recognize inflammation. But it is difficult to identify disease in animal that have no overt clinical signs of disease but yet remain ill. In such cases, the veterinarian must have access to proper laboratory testing to identify occult inflammatory disease.

It has long been known that changes in a select group of constitutive plasma proteins occur in the early stages of the inflammatory process in all animals. This interest is generated by the potential for use of acute phase proteins (APPs) to provide an early and reliable signal to the clinician of the presence of any form of inflammatory disease or inflammation, including malignancies (Kaneko et al., 1997). Fibrinogen is an APP produced by the liver. It functions in the clotting mechanism and plays a significant role in the body defense by moving into extra vascular spaces to assist in localization of disease process. Because of fibrinogen's association with inflammatory conditions, estimation of fibrinogen level has been found useful in evaluation of the inflammatory

responses. In some species, such as ruminants, fibrinogen determination is preferred to an ESR estimation (Coles, 1980) and even better predictor of inflammation than the leukogram ((Kaneko et al., 1997).

The plasma: fibrinogen ration in various clinical entities of the dog, cat, cow and horse has been studied (Schalm, 1970 as cited by Jain, 1986). The plasma fibrinogen levels in normal and sick cow were studied by McSherry et al., 1970; as cited by Coles, 1980). Sutton and Hobman, 1975 as cited by Coles, 1980) studied fibrinogen levels in cattle and compared with total leukocyte and neutrophils count. Some veterinarians believe that the changes in the leukogram alone reflect the inflammatory state in buffalo. Others believe that fibrinogen determination is important.

So, this article tried to study whether fibrinogen alone or in combination of leukogram, is the reliable and better predictor of inflammation. The data from a recent retrospective study of the significance of fibrinogen estimation in various clinical entities of the buffalo, as an adjunct to leukogram interpretation are presented in this research article.

Materials and MethodsTo determine how useful fibrinogen is in detecting inflammation in buffalo, a

retrospective study was conducted at the pathology section of Institute of Agriculture and Animal Sciences (IAAS), Tribhuvan University (TU). The study analyzed 21 sequential leukogram with available fibrinogen data. All samples were from various clinical entities, which were collected from the field during the period 2003 to 2005.

Fibrinogen determination was performed by heat precipitation method and protein determination by refractometer (Coles, 1980). Leukograms were categorized as normal, stress, or inflammatory. Table 1 outlines the criteria that were used for the categorization. Reference intervals of leukogram were derived at pathology department of IAAS(Singh et al,2002).

Stress leukogram in buffalo is generally similar to those reported in cattle (Jain, 1986). The classical stress leukogram include lymhopenia and neutrophilia. I elected to include both of these responses in the stress category; with mild neutrophilia alone, however, underlying mild inflammation cannot be ruled out. For the purposes of this study, an inflammatory leukogram was defined as having one or more of the following characteristics: neutophilia greater than 7000 cells/ml, an increased number of immature neutrophils form, lymphocytosis, monocytosis, or neutropenia.

Corresponding fibrinogen values were categorized as normal (200 to 800 mg/dl) (Jain, 1986) or increased (greater than 800 mg/dl). The fibrinogen values were compared with the classification of the leukogram. Plasma protein/fibrinogen rations were calculated for all samples. Values less than 10 were classified as inflammatory, ration of 10 to 15 were considered to be suggestive of inflammation and ratio greater than 15 were considered normal (Coles, 1980). These data are presented in Table 2.

ResultsOf all leukogram evaluated, 23.81% (5 of 21) were categorize as normal, 9.52%

(2) as stress, and 66.67% ((14) as inflammatory. Of all leukogram, 52.38% (11) had elevated fibrinogen levels.

Of all hemograms associated with normal leukogram, 40% had elevated fibrinogen levels. Of normal leukograms with elevated fibrinogen, 100% had pp/fib rations less than 10, 0% had rations of greater than 10. Of normal leukograms with normal fibrinogen levels, 0% had pp/fib rations less than 10, 66.67% had rations of 10 to 15, and 33.33% had rations greater than 15. Of normal leukograms with increased or normal fibrinogen values, 40% had rations less than 10, 40% had rations of 10 to 15, and 20% had rations greater than 15 (figure 1)

Of all stress leukogram, 50% had elevated fibrinogen. Of stress leukogram with elevated fibrinogen, 100% had pp/fib rations less than 10, 0% had rations greater than 10. Of stress leukogram with normal fibrinogen levels, 0% had rations less than 10, 100% had rations of 10 to 15, and 0% had rations greater than 15. Of stress leukogram with increased or normal fibrinogen values, 50% had rations less than 10, 50% had rations of 10 to 15, and 0% had rations greater than 15.

Of all inflammatory leukograms, 64.3% had elevated fibrinogen. Of inflammatory leukograms with elevated fibrinogen, 88.9% had pp/fib rations less than10, 11.1% had rations of 10 to 15, and 0% had rations greater than 15. Of inflammatory leukograms with normal fibrinogen, 20% had rations less than 10, 40% had rations of 10 to 15, and 40% had ration greater than 15. Of inflammatory leukograms with increased or normal fibrinogen values, 64.3% had rations less than 10, 21.43% had rations of 10 to15, and 7.14% had rations greater than 15.

DiscussionsIn this retrospective study, more patient with normal or stress leukogram had

normal fibrinogen and pp/fib ration of 10 to 15 or greater than 15. Hematologically, there was no evidence of inflammatory disease in these buffalo; however, 42.8% (3 of 7) of all buffalo with stress or normal leukograms had pp/fib rations less than 10, which would indicated inflammation. In these cases, inflammatory disease would not have been identified if the fibrinogen concentration were not determined.

Many factors may contribute to the lack of an inflammatory leukogram in these cases. In the presence of established inflammatory disease, granulopiosis in the bone marrow can reach a steady state so that the demand is met but the white blood cells do not accumulate in the circulation if the leukogram is evaluated during an early transitional stage of neutrophil kinetics, the peripheral blood picture may not reflect the current tissue demand or bone marrow response. In addition, compared to other species, cows have poor granulocytic response early in the disease process (Dina et al, 1994). Another cause may be the degree and cause of inflammatory process.

My findings that 42.8 % of the buffaloes with normal or stress leukograms had evidence of inflammation based on pp/fib ratio is higher to that in a study in horses (Diana et al 1994); and cows (Sutton and Hobmann, 1975 as sited by Coles). Extrapolating from their data, 7.7% of horses and 10% cows with normal leukograms had pp/fib rations less than 10. A comparison of my data and their study suggests that fibrinogen determination in buffaloes is more important than in horse and cows for detecting inflammatory disease. Kaneko et al, 1997 mentioned that in ruminants fibrinogen is the better predictor of inflammation than leukogram.

In the current study, 35.71% (5 of 14) of buffaloes with inflammatory leukograms had no clear evidence of inflammation based on pp/fib rations of 10 to 15 or greater than

15. Khan et al (1997) also studied plasma fibrinogen concentration (Fib), total leukocyte count (TLC), and neutrophil, in buffaloes suffering from different clinical conditions. Authors observed that fibrinogen increased significantly in chronic mastitis, pyrexia, pyometra, cutaneous abscesses, tail gangrene and acute indigestion, whereas in most of the other conditions studied it varied non-significantly. Sutton and Hobman, 1975 (as cited by Coles), also cautioned, that as a sole diagnostic aid, the value of fibrinogen estimation should be treated cautiously. These authors felt that as an indicator of inflammatory process in cattle, fibrinogen estimation should be used as an adjunct to cell counts in detecting necrotic or inflammatory conditions. There are several possible explanations for these observed discrepancies in buffaloes. First, the heat precipitation method for determining fibrinogen may underestimate the actual fibrinogen concentration. Second the value used to define an inflammatory leukogram (greater than 7000 neutrophils/ul) might have been inappropriate; the buffaloes with neutrophils count slightly above 7000 cells/ul had stress leukogram, not inflammatory leukogram. Third, buffaloes with pp/fib ratios of 10 to 15 may have inflammation that is detectable, but other tests and/ or serial evaluations are necessary to confirm it.

In the study, smaller sample size was studied. A similar investigation should be performed using higher number of buffaloes. In addition, comparisons of fibrinogen determination and leukogram interpretations should be performed on specific, naturally occurring and experimentally induced disease process to determine whether fibrinogen values are more important in certain disease state.

ConclusionIt is clear from this study that fibrinogen can be useful in identifying

inflammation in buffaloes if changes in leukogram do not suggest inflammation. By contrast, fibrinogen is not elevated in some cases despite the presence of inflammatory leukogram. Fibrinogen estimation should be used as an adjunct to complete blood count to maximize the chance of detecting necrotic or inflammatory conditions. In addition, serial leukogram and fibrinogen determination may be necessary for hematologic identification of inflammatory disease in buffaloes.

References

Kaneko, J J. Serum protein and dysproteinemias. In Kaneko, J J, Harvey, J W, Bruss M. L. Clinical Biochemistry Of Domestic Animals, 5th ed. Academic press, London, UK, 1997.Coles, EH. Veterinary Clinical Pathology. 3rd ed. WB Saunders Company. pp. 90-91. 1980.Jain, NC. Schalm's Veterinary Hematology. 4th ed. KM Vargheese Company. pp.329. 1986.Diana, AA, Reagan, WJ, DeNicola, DB, 1994. Plasma Fibrinogen in Recognizing Equine Inflammatory Disease. Compendium on Continuing Education for Practicing Veterinarian. vol. 16(10). 1349-1356. Khan ZM, Muhammad G, Umar A, Ali Khan S. 1997. A preliminary comparison of plasma fibrinogen concentrations, leukocyte numbers and erythrocyte sedimentation rate

as non-specific indicators of inflammatory conditions in buffalo (Bubalis bubalis).Vet Res Commun. May;21(4):265-71. Singh DK, Kobayashi K, Furuichi M, and Dhakal IP 2002. The blood Cellular and Serum Profiles of Clinically Healthy Buffaloes in Chitwan, Nepal.Proceedings: 12th FAVA and 14th VAM Congress 2002.

Table 1

Criteria Used to Classify LeukogramsClassificationNormal (all of the following)

Cell TypeNeutrophilsLymphocytesMonocytesEosinophils

Cell count/ul744-33983925-655480-567198-3425

Stress (one or both of the following abnormalities)

LymphocytesNeutrophils

less than 39253400-7000

Inflammatory (one or more of the followings)

NeutrophilsLymphocytesMonocytesNeutrophils

more than 7000more than 6554more than 567less than 744

Table 2Classification of 21 Leukograms Based on Fibrinogen Concentration and Plasma Protein/ fibrinogen ration #.

      Plasma Protein/Fbrinogen ratio           

Leukogram Fibrinogen Concentration Less than 10 10 to 15 Greater than 15

Normal Normal 3(60%) 0(0%) 2(66.67%) 1(33.3%)

Increased 2(40% 2(100%) 0(0%) 0 (0%)

Total 5 2(40%) 2(40%) 1(20%)

Stress Normal 1(50%) 0(0%) 1(100%) 0(0%)

Increased 1(50%) 1(100%) 0(0%) 0(0%)

Total 2 1(50%) 1(50%) 0(0%)

Inflammatory Normal 5(37.7%) 1(20%) 2(40%) 2(40%)

Increased 9(64.3%) 8(88.9%) 1(11.1%) 0(0%)

Total 14 9(64.3%) 3(21.4%) 1(7.14%)           

# Values in parentheses are percentages of the total for that group.

Figure 1 - Classification of plasma protein/fibrinogen ratios in patients with normal, stress, or inflammatory leukograms. Percentages are calculated from the total of each group.